bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin is essential for accumulation of casein mRNA in mouse mammary epithelial cells.
Authors: Bolander F F , Nicholas K R , Van Wyk J J , Topper Y J
Published: May 2006

In the presence of cortisol and prolactin, insulin at concentrations as low as 1 ng/ml significantly stimulates casein synthesis in mammary explants from midpregnant mice; maximal synthesis occurs at 10 ng/ml. However, in the absence of insulin, no detectable immunoprecipitable casein is produced. Insulin also supports enhanced accumulation of casein mRNA in the presence of cortisol and prolactin; neither epidermal growth factor nor somatomedin C has this effect. These inductive actions of insulin are not secondary to a general maintenance effect on the mammary epithelial cell; insulin, epidermal growth factor, and somatomedin C can support the accumulation of RNA in rough endoplasmic reticulum equally well. In addition, these effects do not reflect a specific insulin requirement for prolactin sensitivity; epidermal growth factor can support prolactin-induced total RNA synthesis as well as insulin can. The results demonstrate that, although insulin, epidermal growth factor, and somatomedin C can all function as cell maintenance agents, only insulin, together with cortisol and prolactin, can induce casein mRNA accumulation.
Induction of gene mutation in and cell transformation of mammalian cells by modified purines: 2-aminopurine and 6-N-hydroxylaminopurine.
Authors: Barrett J C
Published: May 2006

2-Aminopurine, a classical mutagen in prokaryotic systems, is inactive as a carcinogen in two animal species. To determine the basis for this discrepancy in the correlation between carcinogenesis and mutagenesis, the ability of 2-aminopurine to induce somatic mutation and neoplastic transformation concomitantly in the same cellular system was examined. 6-N-hydroxylaminopurine, a related modified purine that is a mutagen and a carcinogen, was also studied. 2-Aminopurine was a mutagen in Syrian hamster embryo cells, but its activity was very weak. The maximum induced mutation frequency with either of two mutational markers was only 7 X 10(-6) mutants per surviving cell. 2-Aminopurine also induced morphological transformation of the cells under the same conditions, but the frequency was only approximately 0.04% per surviving colony. Neoplastic transformation of the cells after 2-aminopurine treatment was not observed in these experiments. These results indicate that 2-aminopurine is, at best, a weak transforming agent. The lack of carcinogenic activity in vivo with 2-aminopurine is consistent with these observations. In contrast to the results with 2-aminopurine, 6-N-hydroxylaminopurine was a very effective mutagen in these cells (up to 10(-3) mutants per survivor) and induced morphological transformation of the cells in a dose-dependent manner. Furthermore, neoplastic transformation was induced by this nucleic acid base analog. The correlation of mutagenic activity with transforming ability of these two modified purines supports a relationship between mutagenesis and carcinogenesis. However, relative to other carcinogens, there is a quantitative difference in the ability of 6-N-hydroxylaminopurine to induce cell transformation and mutation. For example, in benzo[a]pyrene-treated cultures, the ratio of the frequency of induced morphological transformation to that of somatic mutation was approximately 100, whereas for 6-N-hydroxylaminopurine-treated cultures, the ratio of transformation to mutation was only 3-12.5. This indicates that 6-N-hydroxylaminopurine is less potent than benzo[a]pyrene in inducing transformation when compared at equal mutagenic potency. This is consistent with our hypothesis that cell transformation, and possibly cancer, occurs predominantly as the result of a mutation at the chromosome level rather than a gene mutation.
Regional gene assignment of human porphobilinogen deaminase and esterase A4 to chromosome 11q23 leads to 11qter.
Authors: Wang A L , Arredondo-Vega F X , Giampietro P F , Smith M , Anderson W F , Desnick R J
Published: May 2006

The regional gene assignments for human porphobilinogen deaminase (PBGD; EC 4.3.1.8) and esterase A4 (ESA4; EC3.1.1.1) chromosome 11 have been determined with somatic cell hybridization and immunologic, electrophoretic, and cytogenetic techniques. Dimethyl sulfoxide-induced erythroid differentiation of hybrid clones derived from the fusion of tetraploid Friend murine erythroleukemia (2S MEL) cells deficient in thymidine kinase and human Lesch--Nyhan fibroblasts (HLN) deficient in hypoxanthine phosphoribosyltransferase (HPRT-; EC 2.4.2.8) were examined for expression of human PBGD, ESA4, and lactate dehydrogenase A (LDHA; EC 1.1.1.27). Human PBGD was detected by rocket immunoelectrophoresis with rabbit anti-human PBGD IgG and by isoelectric focusing. The human chromosome complement of each clone was determined by cytogenetic and enzyme marker analyses. Of the five primary 2S MEL--HLN clones examined, three were positive for human PBGD. These were subcloned to yield a total of 10 secondary, tertiary, or quaternary clones. Analyses of these subclones permitted the regional assignment of human PBGD and ESA4 to the long arm of chromosome 11. Finer regional assignment of the loci for human PBGD and ESA4 was facilitated when two 2S MEL (HPRT-)--human fibroblast (HX/11) hybrids, each containing the X chromosome--autosome translocation (der11), t(X;11)(q25-26;q23) as the only human chromosome, were examined for expression of human PBGD, ESA4, and LDHA. One clone, HX/11-2, contained the intact X/11 translocated chromosome; in the other, HX/11-3, 11p was deleted, and the human X/11 derivative was translocated onto a mouse chromosome. HX/11-2 expressed human LDHA, but HX/11-3 did not, verifying that the latter human 11/X derivative did not include 11pter leads to 11p12; PBGD and ESA4 were not detected in either hybrid. These results confirm the location of the gene for human PBGD on chromosome 11 and establish the assignment of the loci for PBGD and ESA4 in the region 11q23 leads to 11qter.
Mannose 6-, fructose 1-, and fructose 6-phosphates inhibit human natural cell-mediated cytotoxicity.
Authors: Forbes J T , Bretthauer R K , Oeltmann T N
Published: May 2006

In vitro human natural cell-mediated cytotoxicity (NCMC) to K-562, Molt-4, and F-265 cells is inhibited in a dose-dependent manner by mannose 6-phosphate, fructose 1-phosphate and fructose 6-phosphate. This inhibition is not observed with mannose, glucose, fucose, glucose 6-phosphate, mannose 1-phosphate, galactose 1-phosphate, or galactose 6-phosphate. Preincubation of the effector cells, obtained from fresh whole blood, with mannose-6-phosphate, fructose-1-phosphate, or fructose-6-phosphate did not inhibit cytotoxicity, which indicated that these hexose phosphates are not nonspecifically toxic towards the effector lymphocytes. Mannose-6-phosphate and the stereochemically similar fructose-1-phosphate are more potent inhibitors than fructose-6-phosphate in terms of concentration required and time of onset of effect. Inhibition of cytotoxicity by mannose-6-phosphate varied with target cell type: F-265 is protected at much lower concentrations of mannose-6-phosphate (less than 1 mM) than is either Molt-4 or K-562. The inhibition of NCMC is also observed with the inhibitors of lysosomal function, NH4Cl, and chloroquine. The presence of a functional mannose-6-phosphate receptor on target cells was demonstrated: (i) Gelonin, a seed protein that inactivates the eukaryotic ribosome but is nontoxic to intact cells, was covalently linked to monophosphopentamannose, and this conjugate ws toxic to both K-562 and F-265 target cells, the latter being by far the more sensitive; and (ii) chloroquine, NH4Cl, and mannose-6-phosphate all inhibited the toxicity of gelonin-monophosphopentamannose. These results suggest either that a cytolytic lymphokine contains a hexose phosphate residue and may be taken up by target cells through the lysosomal/mannose 6-phosphate pathway or that such a residue is involved in target cell-effector cell recognition.
Ovalocytic erythrocytes from Melanesians are resistant to invasion by malaria parasites in culture.
Authors: Kidson C , Lamont G , Saul A , Nurse G T
Published: May 2006

Ovalocytic erythrocytes from Melanesians in Papua New Guinea have been demonstrated to be resistant to infection by malaria parasites (Plasmodium falciparum) in culture by using a double-label fluorescence assay of merozoite invasion. That merozoites do not bind irreversibly to ovalocytes has been demonstrated by an assay that measures competition between ovalocytes and normocytes. Analysis of behavior on thermal deformation has demonstrated that ovalocytes are more more thermostable than normocytes, suggesting that there is a major difference in cytoskeletal structure. These findings with P. falciparum and epidemiological data demonstrating clinical resistance to P. vivax and P. malariae suggest that the membrane alterations(s) in these ovalocytes affect(s) invasion step(s) common to all three species of malaria parasite.
Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons.
Authors: Goedert M , Stoeckel K , Otten U
Published: May 2006

Nerve growth factor is retrogradely transported in sympathetic and sensory neurons throughout life. Although this transport is known to be biologically significant in sympathetic neurons, such a function was not yet known in sensory ganglia. By using the neuropeptide substance P as a biochemical marker, we show that sensory ganglia from newborn and adult rats respond in nerve growth factor and that its retrograde axonal transport is biologically relevant, as indicated by an increase in substance P and in general protein content.
Nonrandom association of polymorphic restriction sites in the β-globin gene cluster
Authors: Antonarakis Stylianos E. , Boehm Corinne D. , Giardina Patricia J. V. , Kazazian Haig H.
Published: May 2006

By using probes for ε-, Ψβ
1
-, and β-globin genes, we found four additional polymorphic restriction sites that have frequencies >0.1 in persons of Mediterranean area origin, Asian Indians, and American Blacks. Three of these (
Hinc
II sites) and the two previously described polymorphic
Hind
III sites [one in intervening sequence (IVS) II of each γ-globin gene] are distributed over 32 kilobases (kb) of DNA located 5′ to the δ-globin gene. This region of DNA comprises two-thirds of the β-globin gene cluster. Since each of these five polymorphic sites can be present (+) or absent (-), in theory there exist 32 possible combinations of sites (haplotypes). However, in Italians, Greeks, Indians, and Turks, 3 of the 32 haplotypes, (+----), (-+-++), and (-++-+), account for 92% of 89 β
A
chromosomes examined. The observed frequencies for these haplotypes are 0.64, 0.15, and 0.13 in the populations studied, in contrast to expected frequencies (based on the observed gene frequencies at each of the five sites) of 0.20, 0.006, and 0.005, respectively. In American Blacks, a fourth haplotype, (----+), which is rare in non-Black populations, has a frequency of 0.37 in contrast to its expected frequency of 0.05. These results suggest a nonrandom association of DNA sequences over 32 kb 5′ to the δ-globin gene in all populations studied. Two other polymorphic sites 3′ to the δ gene (the newly discovered
Ava
II site in IVS II of the β-globin gene and the
Bam
HI site 3′ to it) are nonrandomly associated with each other but randomly distributed with respect to the above haplotypes. This suggests that randomization of sequences has occurred within 12 kb of DNA between these two nonrandomly associated sequence clusters. Nonrandom association of polymorphic restriction sites has practical consequences in that it limits the usefulness of these additional
Hinc
II sites for prenatal diagnosis of hemoglobinopathies by linkage analysis. These sites provide little additional information for detection of β-thalassemia, while the polymorphic
Ava
II site, which lies outside the nonrandomly associated sequences 5′ to the δ gene, improves the test applicability from 52% to 70% of couples at risk.
In vivo regulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase: immunotitration of the enzyme after short-term mevalonate or cholesterol feeding.
Authors: Arebalo R E , Tormanen C D , Hardgrave J E , Noland B J , Scallen T J
Published: May 2006

In recent studies using either a single dose of mevalonolactone administered by intragastric tube or a single meal containing 2% cholesterol, it was demonstrated that rat liver hydroxymethylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] (HMG-CoA reductase) the major regulatory enzyme in cholesterol biosynthesis, is subject to two phases of inhibition. The first phase of inhibition is explained by in vivo phosphorylation of the enzyme; however, the nature of the second phase of inhibition remained obscure. The present study tested two possible explanations for this second phase of inhibition--increased enzyme turnover leading to a decreased concentration of HMG-CoA reductase molecules, and further inactivation of existing enzyme molecules. The results with the technique of immunotitration of HMG-CoA reductase show that, in short-term studies conducted up to 2 hr after the administration of a single dose of mevalonolactone or up to 6 hr after a single meal of rat chow containing 2% cholesterol, the in vivo regulation of rat liver HMG-CoA reductase during the first half of the dark period does not occur by increased enzyme turnover but, instead, existing enzyme is further inactivated.
Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of
Agrobacterium tumefaciens
Authors: Murai Norimoto , Kemp John D.
Published: May 2006

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of
agrobacterium tumefaciens
and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated
in vitro
in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of
M
r
40,000 (the same as authentic octopine synthase) was synthesized
in vitro
by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five
32
P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the
M
r
40,000 polypeptide and was not detected in the habituated callus.
Gross quaternary changes in aspartate carbamoyltransferase are induced by the binding of N-(phosphonacetyl)-L-aspartate: A 3.5-A resolution study.
Authors: Ladner J E , Kitchell J P , Honzatko R B , Ke H M , Volz K W , Kalb A J , Ladner R C , Lipscomb W N
Published: May 2006

The three-dimensional structure of the complex of N-(phosphonacetyl)-L-aspartate with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been determined to a nominal resolution of 3.5 A by single-crystal x-ray diffraction methods. Initial phases were obtained by the method of "molecular tectonics": beginning with the structure of the CTP-protein complex, the domains of the catalytic and regulatory chains were manipulated as separate rigid bodies. The resulting coordinates were used to calculate an electron density map, which was then back transformed to give a set of calculated amplitudes and phases. Using all observed data, we obtained a crystallographic R factor between observed and calculated amplitudes Fo and Fc of 0.46. An envelope was then applied to a 2Fo - Fc map and the density was averaged across the molecular twofold axis. Two cycles of averaging yielded an R factor of 0.25. In this complex, we find that the two catalytic trimers have separated from each other along the threefold axis by 11-12 A and have rotated in opposing directions around the threefold axis such that the total relative reorientation is 8-9 degrees. This rotation places the trimers in a more nearly eclipsed configuration. In addition, two domains in a single catalytic chain have changed slightly their spatial relationship to each other. Finally, the two chains of one regulatory dimer have rotated 14-15 degrees around the twofold axis, and the Zn domains have separated from each other by 4 A along the threefold axis. These movements enlarge the central cavity of the molecule and allow increased accessibility to this cavity through the six channels from the exterior surface of the enzyme.
Nonadenylylated mRNA is present as polyadenylylated RNA in nuclei of Drosophila.
Authors: Zimmerman J L , Fouts D L , Levy L S , Manning J E
Published: May 2006

The sequence complexity of nuclear total RNA and nuclear poly(A)+RNA from Drosophila third-instar larvae was determined by hybridization of these RNAs to labeled single-copy DNA. At saturation, the nuclear poly(A)+ - and total RNA hybridized to 11% and 22.5% of the single-copy DNA, respectively. The increase in complexity of nuclear total RNA over that observed for nuclear poly(A)+RNA indicates the presence of a discrete class of nonoadenylylated nuclear RNA molecules. The relationship between DNA sequences coding for nuclear RNA and mRNA was then determined by hybridization of nuclear total and poly(A)+RNA to DNA enriched for mRNA coding sequences. The results of these studies show that those single-copy DNA sequences that are represented in either the poly(A)+ - or poly(A)- mRNA population are transcribed into RNA molecules that appear in the nuclear poly(A)+RNA population.
Molecular evolution, intracellular organization, and the quinary structure of proteins.
Authors: McConkey E H
Published: May 2006

High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most polypeptides participate. It is suggested that the term "quinary structure" be used to refer to macromolecular interactions that are transient in vivo. Such interactions will not be evident from the composition of purified proteins, but they may constitute an important source of constraints on changes in primary structure.
Novel features of animal mtDNA evolution as shown by sequences of two rat cytochrome oxidase subunit II genes.
Authors: Brown G G , Simpson M V
Published: May 2006

The sequence of the region of the mitochondrial genome that encodes cytochrome oxidase subunit II (COII) has been determined for each of two closely related rat species, Rattus norvegicus and R. rattus. Comparison of the two sequences shows that 94.4% of the nucleotide substitutions are silent. The occurrence of this high proportion of silent substitutions leads us to propose that the rapid evolution of mtDNA relative to nuclear DNA is due only to silent changes and that amino acid-altering substitutions accumulate in nuclear and mtDNA at comparable rates. Other novel features of the nucleotide substitution pattern in the rat COII gene are a high transition/transversion ratio (8.0:1) and a strong bias toward C in equilibrium T transitions in the light strand. Comparison of the R. norvegicus COII sequence with the bovine and human sequences show that there may be selective constraints on some silent positions within the gene and that its rate of evolution may be different in different mammalian lineages.
Polymorphism in immunoglobulin heavy chains suggesting gene conversion.
Authors: Clarke S H , Claflin J L , Rudikoff S
Published: May 2006

Complete heavy (H) chain variable region (V region; amino acids 1-118) sequences have been determined for three phosphocholine (PCho)-binding monoclonal antibodies of CBA mouse strain origin. Two of these were found to differ from the sequence of the BALB/c T15 germline VH segment (segment of the V region that includes amino acids 1-95) at four positions but were identical to the allelic form of T15 (C3) found in C57BL. The third VH segment, HP101.6G6 (6G6), was clearly the product of a second, related VH gene, probably the allele of the BALB/c V11 gene, a second member of the P-Cho VH gene family. Thus, more than one VH gene is capable of encoding heavy chains of PCho-binding antibodies. The 6G6 VH segment differs from VII at seven positions; four of these distinguishing amino acids are encoded in other membranes of the PCho VH gene family. We postulate that the origin of the 6G6 VH sequence can most easily be explained by a process of gene conversion occurring between the least three members of the PCho VH family.
Modulation of T-lymphocyte differentiation antigens: potential relevance for multiple sclerosis.
Authors: Antel J , Oger J J , Jackevicius S , Kuo H H , Arnason B G
Published: May 2006

Effects of the anti-T-cell monoclonal antibodies OKT3, OKT5, and OKT8 on T-cell surface properties and cell functions were evaluated. Incubation of mononuclear cells isolated from peripheral blood for 48 hr with each monoclonal antibody in the absence of complement resulted in modulation of their respective surface antigens; i.e., the number of cells detected by immunofluorescence as positive for the T3, T5, and T8 surface antigens was reduced. T3, T5, and T8 antigens modulated independently. A radiolabeled second antibody technique confirmed modulation by OKT3 and OKT8 and indicated that T-cell differentiation antigens can regenerate in culture. Incubation of mononuclear cells with OKT3 increased the number of sheep erythrocyte-binding lymphocytes (E+-rosetting cells) and markedly increased the number of avidly E+-rosetting cells. Incubation with OKT8 reduced the number of E+- and of avidly E+-rosetting cells. OKT3 induced both mitogenic reactivity and suppressor cell activity; cells modulated by OKT8 exhibited reduced mitogenic reactivity and reduced suppressor cell function. The decreases in total T cells, in avid T cells, in suppressor cell number, and in suppressor cell function that follow modulation by OKT8 mimic changes observed in multiple sclerosis patients.
Interaction of the Escherichia coli dnaA initiation protein with the dnaZ polymerization protein in vivo.
Authors: Walker J R , Ramsey J A , Haldenwang W G
Published: May 2006

To define in vivo interactions of Escherichia coli DNA replication components, extragenic suppressors of a dnaZ(TS) mutant were isolated. A temperature-sensitive dnaZ mutant, which is defective in polymerization, was placed at 39 degrees C to select temperature-insensitive revertants. Some of these revertants also were cold sensitive, a phenotypic property that facilitated study of the suppressor. Mapping of the cold sensitivity indicated that some of the suppressor mutations are intragenic but others are located within the initiation gene, dnaA. The dnaA mutations that suppress the dnaZ(TS) defect are designated dnaA(SUZ, CS). The dnaA(SUZ, CS) strains have a defect in DNA synthesis at low temperature that is typical of an initiation defect. These data suggest that the dnaA product, an initiation factor, interacts in vivo with the dnaZ protein, a polymerization factor.
Regional cerebral glucose utilization during morphine withdrawal in the rat.
Authors: Wooten G F , DiStefano P , Collins R C
Published: May 2006

Regional cerebral glucose utilization was studied by 2-deoxy[14C]glucose autoradiography in morphine-dependent rats and during naloxone-induced morphine withdrawal. In morphine-dependent rats, glucose utilization was increased compared with naive controls uniformly (23-54%) in hippocampus, dentate gyrus, and subiculum and reduced in frontal cortex, striatum, anterior ventral thalamus, and medial habenular nucleus. On precipitation of morphine withdrawal by subcutaneous administration of naloxone at 0.5 mg/kg to morphine-dependent rats, glucose utilization was increased in the central nucleus of amygdala (51%), lateral mammillary nucleus (40%), lateral habenular nucleus (39%), medial mammillary nucleus (35%), and medial septal nucleus (35%) (all, P less than 0.01). Significant increases also occurred in several other limbic structures including interpeduncular nucleus, anterior medial and ventral thalamic nuclei, and lateral septal nucleus. Knowledge of the functional cerebral anatomy of the morphine-withdrawal syndrome should facilitate studies directed toward understanding the molecular mechanisms of opiate withdrawal.
Stimulation of cyclic AMP accumulation and corticotropin release by synthetic ovine corticotropin-releasing factor in rat anterior pituitary cells: site of glucocorticoid action.
Authors: Giguère V , Labrie F , Côté J , Coy D H , Sueiras-Diaz J , Schally A V
Published: May 2006

A 2.5-fold stimulation of cyclic AMP cellular content is measured 60 sec after addition of 100 nM synthetic ovine corticotropin-releasing factor (C-RF; corticoliberin) to rat anterior pituitary cells in culture. A maximal response of cyclic AMP content at 400% above control is observed between 2 and 30 min after addition of the peptide, whereas an 8-fold stimulation of cyclic AMP released into the incubation medium is measured between 10 and 180 min. A linear 7-fold increase of corticotropin release is observed for up to 3 hr. Preincubation from 18 hr with the potent glucocorticoid dexamethasone has no effect on C-RF-induced cyclic AMP accumulation. The same treatment with dexamethasone causes an 80% inhibition of corticotropin release induced by both C-RF and the cyclic AMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate. The present data show that ovine C-RF is a potent stimulator of cyclic AMP accumulation in rat anterior pituitary cells and that the process is insensitive to the action of dexamethasone. The marked inhibition by dexamethasone of corticotropin secretion induced by a cyclic AMP derivative indicates that glucocorticoids exert their potent inhibitory effects on corticotropin secretion at a step distant to cyclic AMP formation.
Analysis of type 5 adenovirus transformation with a cloned rat embryo cell line (CREF).
Authors: Fisher P B , Babiss L E , Weinstein I B , Ginsberg H S
Published: May 2006

A recently isolated cloned cell line of Fischer rat embryo fibroblasts (CREF) can be transformed at high frequency by adenovirus serotype 5 (Ad5). The CREF cells display a near diploid karyotype, do not crisscross at confluency, can be maintained at confluency for greater than 7 wk at 36 degrees C, and do not form macroscopic colonies when seeded in agar. Transformed cells are identified by their altered morphology and the presence of adenovirus DNA sequences in the transformants, which can be demonstrated by lysing cells directly on nitrocellulose filters and hybridizing with 32P-labeled Ad5 DNA (spot hybridization). The frequency of transformation at 36 degrees C is approximately equal to 2 x 10(-4) with wild-type Ad5 and approximately equal to 2 x 10(-3) with the temperature-sensitive mutant H5ts125. Southern blot hybridization analysis indicates that five out of six wild-type Ad5- and six out of six H5ts125-transformed CREF clones isolated at 36 degrees C contain the complete integrated Ad5 genome. Preliminary characterization of four transformed clones (two wild-type and two H5ts125) indicates that, even though transformation was done in CREF cells (a clonal cell line), they differ in their biological properties, such as saturation density and anchorage dependence.
Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.
Authors: Shultz T D , Bollman S , Kumar R
Published: May 2006

The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ligated loop technique revealed a 60% increase in calcium absorption compared to control birds (P less than or equal to 0.001). However, the administration of cortisol in various doses (3 and 5 mg) to chickens given 1,25-(OH)2D3 resulted in significant decreases in intestinal calcium transport in vivo (P less than or equal to 0.05; P less than or equal to 0.05). When intestinal BBM vesicles were prepared from birds treated in a manner identical with that described above, there was no observable difference between calcium uptake in BBM vesicles of the 1,25-(OH)2D3-treated birds and that of the cortisol plus 1,25-(OH)2D3-treated birds. 1,25-(OH)2D3-treated and 1,25-(OH)2D3 plus cortisol-treated chicks had intestinal BBM vesicle uptakes that were significantly greater than those of vitamin D-deficient controls (P less than or equal to 0.02; P less than or equal to 0.025). These data show that in vivo intestinal calcium transport may be markedly reduced in the presence of normal intestinal BBM vesicle calcium uptake. This suggest that factors other than BBM calcium uptake (e.g., protein synthesis or contraluminal membrane events) play an important role in the movement of calcium from the intestinal lumen into the bloodstream and extracellular fluid of the organism.
Long axons within the striate cortex: their distribution, orientation, and patterns of connection.
Authors: Mitchison G , Crick F
Published: May 2006

Rockland and Lung [Rockland, K. S. & Lung, J. S. (1982) Science 215, 1532-1534] have recently observed that an injection of horseradish peroxidase into the striate cortex of the tree shrew produces a patchy distribution of label adjacent to the injection site. They proposed that this pattern might be due to populations of neurons with long-range cortico-cortical connections that are interspersed with populations having no such connections. We suggest here an alternative explanation. We can account for the pattern by supposing that the label is carrier by a system of oriented axons. We suppose that these axons link cells with similar orientation preferences and make their connections within a narrow strip of cortex whose direction is related to the orientation of the cells in question. We suggest that such connections could be involved in generating complex receptive fields from simple ones. Other possibilities are that they are used to generate very elongated receptive fields, inhibitory flanks, or end-stopping. We suggest a number of experimental tests of these ideas.
Theory of periodic structures in lipid bilayer membranes
Authors: Falkovitz Meira S. , Seul Michael , Frisch Harry L. , McConnell Harden M.
Published: May 2006

An approximate, new model for the structure of the periodic, undulated
P
β′
, phase of phosphatidylcholine bilayers is proposed. The properties of this phase are deduced by minimizing a Landau-de Gennes expression for the bilayer free energy when this free energy contains a term favoring a spontaneous curvature of the membrane. The theoretical calculation leads to a model for the
P
β′
phase of phosphatidylcholine bilayers having a number of novel physical properties, including periodic variations in membrane “fluidity.”
Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.
Authors: Pettit F H , Humphreys J , Reed L J
Published: May 2006

Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffective. Hydrogen peroxide and t-butyl hydroperoxide were inactive. The data indicate pyruvate dehydrogenase kinase (EC 2.7.1.99) contains a thiol group (or groups) that is involved in maintaining a conformation of the enzyme that facilitates phosphorylation and inactivation of its protein substrate, pyruvate dehydrogenase (EC 1.2.4.1). These findings suggest that modulation of pyruvate dehydrogenase kinase activity by thiol-disulfide exchange may be an important physiological mechanism for regulation of kinase activity and, hence, activity of the pyruvate dehydrogenase complex.
Ethylene regulation of gene expression in carrots
Authors: Christoffersen Rolf E. , Laties George G.
Published: May 2006

The respiration of carrot (
Daucus carota
L.) roots is stimulated by ethylene. Polyribosomes were shown to proliferate concomitantly with the increase in respiration, and the extent of polyribosome augmentation was closely correlated to the amount of respiratory stimulation. In addition to the increase in quantity, ethylene caused a 2-fold increase in the average polyribosome size, suggesting tighter packing of ribosomes on RNA.
In vitro
translation of carrot polyadenylylated RNA with the rabbit reticulocyte lysate system followed by electrophoresis of the resulting translation products showed that ethylene treatment results in the appearance of new mRNAs.
Internalization and molecular processing of insulin receptors in isolated rat adipocytes.
Authors: Berhanu P , Olefsky J M , Tsai P , Thamm P , Saunders D , Brandenburg D
Published: May 2006

The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with 125I-labeled NAPA-DP-insulin were identified by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands of Mr 330,000, 295,000, and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16 degrees C were immediately trypsinized, all of the receptor bands were degraded into lower molecular weight fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 37 degrees C for 1 hr prior to trypsin exposure, approximately equal to 30% of the receptors were found to be trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37 degrees C (but not at 16 degrees C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16 degrees C and during the subsequent incubation at 37 degrees C showed that this agent (i) increased the trypsin-insensitive (intracellular) receptor pool, (ii) blocked conversion of the Mr 125,000 subunit into the Mr 115,000 component, and (iii) prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulin-receptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).
De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.
Authors: Stewart C L , Stuhlmann H , Jähner D , Jaenisch R
Published: May 2006

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.
Development and maturation of central nervous system myelin: Comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes
Authors: Hartman Boyd K. , Agrawal Harish C. , Agrawal Daya , Kalmbach Sandra
Published: May 2006

The immunohistochemical localization of two myelin specific proteins—basic protein (BP) and proteolipid protein (PLP)—was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (
i
) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (
ii
) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (
iii
) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (
i
) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (
ii
) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (
iii
) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.
Purification and characterization of cysteic acid and cysteine sulfinic acid decarboxylase and L-glutamate decarboxylase from bovine brain.
Authors: Wu J Y
Published: May 2006

L-Cysteic and cysteine sulfinic acids decarboxylase (CADCase/CSADCase) and L-glutamic acid decarboxylase (GADCase), the synthetic enzymes for taurine and gamma-aminobutyric acid, respectively, have been purified to homogeneity from bovine brain. Although CADCase/CSADCase and GADCase copurified through various column procedures, these two enzymes can be clearly separated by a hydroxyapatite column. The purification procedures involve ammonium sulfate fractionation, column chromatographies on Sephadex G-200, hydroxyapatite, DEAE-cellulose, and preparative polyacrylamide gel electrophoresis. The Km values for CADCase/CSADCase are 0.22 and 0.18 mM with L-cysteic and cysteine sulfinic acids as substrates, respectively. CADCase/CSADCase cannot use L-glutamate as substrate. GADCase can use L-glutamate, L-cysteic, and cysteine sulfinic acid as substrates with Km values of 1.6, 5.4, and 5.2 mM, respectively. Antibodies against CADCase/CSADCase do not crossreact with GADCase preparations and vice versa. It is concluded that CADCase/CSADCase and GADCase are two distinct enzyme entities and they are responsible for the biosynthesis of taurine and gamma-aminobutyric acid, respectively.
Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes.
Authors: Nishimura J , Huang J S , Deuel T F
Published: May 2006

Platelet-derived growth factor (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal growth factor (EGF), but not fibroblast growth factor (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is 15 microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell growth stimulated by these separate factors.
A special fructose bisphosphate functions as a cytoplasmic regulatory metabolite in green leaves
Authors: Cséke Csaba , Weeden Norman F. , Buchanan Bob B. , Uyeda Kosaku
Published: May 2006

Fructose 2,6-bisphosphate (Fru-2,6-
P
2
), a regulatory metabolite discovered in animal cells and recently reported to occur in etiolated seedlings, was found to be present in the cytoplasmic fraction of leaves of spinach and peas (typical C
3
plants, in which a three-carbon carboxylic acid is a major early photosynthetic product). At concentrations approximating those calculated to occur physiologically, Fru-2,6-
P
2
modulated two enzymes of the leaf cytoplasm: (
i
) Fructose-1,6-bisphosphatase (EC 3.1.3.11), a key enzyme of sugar synthesis, was competitively inhibited by Fru-2,6-
P
2
, and (
ii
) pyrophosphate-linked phosphofructokinase (inorganic pyrophosphate-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.90), a cytoplasmic enzyme that now seems important in glycolysis of C
3
plants, was activated by Fru-2,6-
P
2
. There was no indication of a role for Fru-2,6-
P
2
in photosynthesis of either chloroplasts or oxygenic prokaryotes. The results suggest that Fru-2,6-
P
2
functions in the regulation of glycolysis and gluconeogenesis (carbohydrate synthesis) in the cytoplasm of leaves of C
3
plants.
Luteolysis-induced changes in phase composition and fluidity of bovine luteal cell membranes.
Authors: Goodsaid-Zalduondo F , Rintoul D A , Carlson J C , Hansel W
Published: May 2006

X-ray diffraction, fluorescence polarization of trans-parinaric acid, and fluorescence photobleaching recovery of dioctadecyltrimethyneindolecarbocyanine have been used to characterize the phase composition and liquid phase fluidity of bovine luteal cell membranes and membrane lipids for functional corpora lutea collected at midcycle and for regressing corpora lutea collected after treatment with prostaglandin F2 alpha. These results support previous observations of gel phases in microsomal preparations of regressed luteal cells at physiological temperatures and further suggest that the plasma membrane may be the main source of this gel phase. Analysis of the overall lipid composition of the microsomal preparations from these cells indicates a role for sphingomyelin, in the presence of cholesterol, for the generation of a gel phase at physiological temperatures.
Evidence for a respiratory chain in the chloroplast
Authors: Bennoun Pierre
Published: May 2006

Evidence is given for the existence of an electron transport pathway to oxygen in the thylakoid membranes of chloroplasts (chlororespiration). Plastoquinone is shown to be a redox carrier common to both photosynthetic and chlororespiratory pathways. It is shown that, in dark-adapted chloroplasts, an electrochemical gradient is built up across the thylakoid membrane by transfer of electrons through the chlororespiratory chain as well as by reverse functioning of the chloroplast ATPases. It is proposed that these mechanisms ensure recycling of the ATP and NAD(P)H generated by the glycolytic pathway converting starch into triose phosphates. Chlororespiration is thus an O
2
-uptake process distinct from photorespiration and the Mehler reaction. The evolutionary significance of chlororespiration is discussed.
BASILEA rabbits express two types of immunoglobulin light chains: lambda and kappa-like.
Authors: Garcia I , Brandt D C , Benammar A , Cazenave P A , Jaton J C
Published: May 2006

In the variant rabbit strain BASILEA, immunoglobulin G were shown to contain two distinct populations of IgG molecules whose light (L) chains belonged to the known lambda isotype and to a new kappa-like type. These two L chains differed from each other by electrophoretic, chemical, and antigenic properties. The kappa-like L chain fraction showed (i) an acid-labile Asp-Pro bond at the end of the joining region and (ii) a tryptic peptide, whose amino acid sequence of the NH2-terminal 15 residues was identical to the homologous constant (C) region sequence of b9 kappa chain with the exception of the residue in position 70, which is asparagine in the kappa-like chain instead of the characteristic half-cystine residue in all L chains of kappa B type expressing b4, b5, b6, or b9 allotypes. The data suggest that the kappa-like L chain component does not contain the C region half-cystine residue involved in the formation of the extra variable (V) region-C region disulfide bridge in L chains of the kappa B [Rejnek, J., Appella, E., Mage, R. G. & Reisfeld, R. A. (1969) Biochemistry 8, 2712-2718]. The partial NH2-terminal amino acid sequence of the C region of the kappa-like L chain was shown to be markedly different from b4, b6, or b9 region sequences and from rabbit lambda C region sequence. Taken together, the chemical data suggest that the kappa bas component represents a new subtype of kappa chain. A rabbit alloantiserum made against bas IgG and adsorbed with IgG fractions showing b4, b5, b6, b95, and b96 L chain allotypes appeared to be directed against the kappa-like L chain component of BASILEA IgG exclusively. All BASILEA animals expressed IgG molecules containing kappa-like chains; in contrast, IgG molecules derived from the standard domestic rabbit did not react with this antiserum.