bluuebunny/crossref_metadata_2025 huggingface dataset:
Early epochal maps of two different cell adhesion molecules.
Authors: Edelman G M , Gallin W J , Delouvée A , Cunningham B A , Thiery J P
Published: May 2006

N-CAM, the neural cell-adhesion molecule, has previously been found to be expressed during several epochs of development and function, first as an early marker in embryo-genesis, later during organogenesis, and finally in adult life. L-CAM, the liver cell-adhesion molecule, has now been localized in embryonic and adult tissues of the chicken by fluorescent antibody techniques. In the early embryonic epoch, L-CAM and N-CAM appeared in epiblastic and hypoblastic tissues. L-CAM was distributed thereafter across all three germ layers. By the onset of neurulation, however, L-CAM disappeared in the region of the neural plate and N-CAM increased in amount in that region. L-CAM appeared strongly on all budding endodermal structures (liver, pancreas, lung, thyroid, parathyroid, thymus, and bursa of Fabricius) whereas N-CAM appeared most strongly in the neural plate, neural tube, and in cardiac mesoderm but was not found in endodermal derivatives. In placodes, both L-CAM and N-CAM were present until the formation of definitive neural structures, at which time L-CAM disappeared. In kidney precursors, the two CAMs followed a complex reciprocal pattern of appearance and disappearance. For the most part, however, the distributions of the two molecules did not overlap during organogenesis. Like N-CAM, L-CAM persisted in a distinctive pattern of expression in adult tissues. During embryonic development, the two different CAMs were distributed on tissues derived from more than two-thirds of the early embryonic surface. Interpretation of maps summarizing CAM distributions over a defined developmental epoch suggested a key role for both L-CAM and N-CAM in embryonic induction. Consistent with this interpretation and with the fact that the continuity of germ layers is lost when organ rudiments are formed, neither of the CAMs was limited in distribution to a single germ layer. The regions of the early epochal maps that lacked both L-CAM and N-CAM comprised some portions of the splanchnopleure and somatopleure. Certain adult tissues that derive from this lateral plate mesoderm such as smooth muscle also lacked L-CAM and N-CAM. Such observations suggest that at least one more CAM may exist in these and similarly derived tissues.
Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.
Authors: Weinstock G M , ap Rhys C , Berman M L , Hampar B , Jackson D , Silhavy T J , Weisemann J , Zweig M
Published: May 2006

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.
Polymorphic gene for human carbonic anhydrase II: a molecular disease marker located on chromosome 8.
Authors: Venta P J , Shows T B , Curtis P J , Tashian R E
Published: May 2006

A panel of 28 mouse-human somatic cell hybrids of known karyotype was screened for the presence of the human carbonic anhydrase II (CA II) gene, which encodes one of the three well-characterized, genetically distinct carbonic anhydrase isozymes (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1). The human and mouse CA II genes can be clearly distinguished by Southern blot analysis of BamHI-digested genomic DNA with a mouse CA II cDNA hybridization probe. The two major hybridizing fragments in mouse were 15 and 6.0 kilobase pairs, and in human they were 15 and 4.3 kilobase pairs. Analysis of the somatic cell hybrids by this technique identified those containing human CA II gene sequences. Segregation analysis of the molecular marker and chromosomes in cell hybrids indicated a clear correlation between the presence of chromosome 8 and the human CA II gene (CA2). This finding provides the second polymorphic marker for human chromosome 8 and, moreover, a molecular disease marker, because human CA II deficiency has recently been linked to an autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification.
Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.
Authors: Gurevitz M , Jain S K , Apirion D
Published: May 2006

A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene. The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature. The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop. It can be processed in vitro to a molecule the size of 10Sb RNA. None of the four endoribonucleases of E. coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction. Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease. The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E. coli cell.
Rearrangement and expression of immunoglobulin genes and expression of Tac antigen in hairy cell leukemia.
Authors: Korsmeyer S J , Greene W C , Cossman J , Hsu S M , Jensen J P , Neckers L M , Marshall S L , Bakhshi A , Depper J M , Leonard W J , Jaffe E S , Waldmann T A
Published: May 2006

The origin and exact stage of differentiation of the neoplastic cells that comprise hairy cell leukemia have remained uncertain. As Ig heavy and light chain genes must both undergo a DNA rearrangement during B-cell development but rarely do so within other hematopoietic lineages, we examined these genes in this leukemia. The neoplastic cells of all eight cases demonstrated rearranged heavy and light chain genes and, in two cases examined, contained the corresponding mRNA for heavy and light chain Ig. Consistent with this B-cell genotype, all cases displayed cell surface HLA-DR and B-cell-associated antigens. Unexpectedly, all cases demonstrated cell surface Tac antigen, which previously had been restricted predominantly to select T-cell malignancies and activated T cells. Prior studies suggested that the anti-Tac monoclonal antibody recognized a peptide associated with the binding of interleukin 2 (T-cell growth factor) in such T cells. Immunoprecipitation with anti-Tac and NaDodSO4/polyacrylamide gel electrophoresis revealed an antigen on leukemic hairy cells with a Mr of 53,000-57,000, identical in size to the receptor on activated T cells. This apparent biphenotypic status might reflect a transformation-associated expression of the Tac antigen in this leukemia. Alternatively, hairy cell leukemia may be a malignancy of a unique stage of normal B-cell differentiation in which the Tac antigen is expressed.
Recognition of female kin by male bees through olfactory signals
Authors: Smith Brian H.
Published: May 2006

After males of
Lasioglossum zephyrum
(Hymenoptera: Halictidae) have first been exposed to a conspecific female, the attractiveness of a second female diminishes with increasing genealogical relationship between the two females. The most parsimonious explanation for this is a polygenically controlled female sex pheromone. Nonrandom mating preferences may markedly affect gene flow within and between natural populations. Polygenically controlled sex pheromones probably also enhance kin recognition among females and, thus, influence the evolution of eusocial behavior in bees and wasps.
Methyl-directed repair of DNA base-pair mismatches in vitro.
Authors: Lu A L , Clark S , Modrich P
Published: May 2006

An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli. The method relies on repair of heteroduplex molecules of f1 R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule. As observed with mismatch heteroduplexes of lambda DNA [Pukkila, P. J., Peterson, J., Herman, G., Modrich, P. & Meselson, M. (1983) Genetics, in press], in vivo mismatch correction of f1 heteroduplexes is directed by the state of dam methylation of d(G-A-T-C) sequences within the DNA duplex. Thus, the heteroduplex (formula: see book) is repaired in vivo to an EcoRI-sensitive form if the strand bearing the wild-type EcoRI sequence carries the dam modification and the other does not. Such molecules are also subject to mismatch repair by E. coli extracts. The in vitro activity is also dependent on ATP, the state of dam methylation of mismatch heteroduplexes, and products of mutH, mutL, mutS, and uvrE loci. However, crude fractions deficient in these gene products do complement in the cell-free system, thus providing assays for their isolation. The in vitro reaction is accompanied by repair synthesis on the unmethylated DNA strand.
Mössbauer and EPR studies of activated aconitase: development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate.
Authors: Emptage M H , Kent T A , Kennedy M C , Beinert H , Münck E
Published: May 2006

During activation of aconitase a ferrous ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or 56Fe for activation we have studied with Mössbauer spectroscopy the beef heart enzyme in the presence of citrate. Our studies show that the environment of one iron site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the iron acquired during activation of aconitase. In the oxidized [4Fe-4S]2+ state two species with enzyme-bound substrate are observed, whereas only one is observed in the reduced [4Fe-4S]+ state. The Mössbauer parameters of Fea reveal that the site has acquired substantial high-spin ferrous character. This is most pronounced in the 1+ state where at Fea the cluster exhibits a localized valence state. The dramatic increase of the isomer shift upon substrate binding strongly suggests that the ligand environment of Fea has become at least five-coordinate and that the cluster may function as a Lewis acid. In the absence of citrate the EPR spectra of the active [4Fe-4S]+ enzyme (g1,2,3 = 2.06, 1.93, 1.86) show no hyperfine broadening in the presence of H2 17O. However, in the presence of citrate (g1,2,3 = 2.04, 1.85, 1.78) sizable transferred hyperfine interactions are observed; under the experimental conditions the hydroxyl groups of citrate and isocitrate as well as water are labeled with 17O. We did not detect broadening by 17O-labeled carboxyl groups of citrate in H2 16O. Implications for the mechanism of aconitase are discussed.
Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor.
Authors: Marquardt H , Hunkapiller M W , Hood L E , Twardzik D R , De Larco J E , Stephenson J R , Todaro G J
Published: May 2006

Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.
Increased membrane permeability to chloride in Duchenne muscular dystrophy fibroblasts and its relationship to muscle function.
Authors: Pato C N , Davis M H , Doughty M J , Bryant S H , Gruenstein E
Published: May 2006

Previous studies have suggested an abnormality in Cl- metabolism in Duchenne muscular dystrophy (DMD) fibroblasts. In order to further characterize this abnormality, we have studied 36Cl- distribution and permeability in 11 DMD and 12 normal fibroblast lines. Under steady-state conditions Cl- efflux in fibroblasts is observed to be biphasic, revealing the presence of two major subcellular compartments. Each compartment contains approximately half of the cellular Cl-. The faster of the two observed efflux components is significantly higher in DMD than in control fibroblasts (P less than 0.001). To determine the results of a similar increase in Cl- permeability on skeletal muscle action potentials, we have simulated the effects of increased Cl- conductance on muscle by using a computer model. Effects on the simulated action potential include lower rates of membrane depolarization, lower overpotential, longer duration, and lower input resistance. These effects are similar to those actually observed in DMD muscle.
Stimulus-response coupling in sponge cell aggregation: Evidence for calcium as an intracellular messenger
Authors: Dunham Philip , Anderson Cathleen , Rich Abby M. , Weissmann Gerald
Published: May 2006

Aggregation of dissociated sponge cells has been proposed as a model for cell-cell recognition mediated by a specific proteoglycan aggregation factor (
Microciona
aggregation factor). To test whether sponge cells undergo stimulus-response coupling in which intracellular Ca is a messenger, aggregation of mechanically dissociated cells was studied. Changes in light transmission through cell suspensions paralleled aggregation as judged by microscopy. In the presence, but not absence, of Ca (>5 mM) partially purified
Microciona
aggregation factor aggregated both living and glutaraldehyde-fixed cells. Evidence for a messenger role of Ca was the following: (
i
) Addition of Ca to Ca-depleted cells induced aggregation that varied with [Ca]. (
ii
) Addition of Ca ionophores (A23187 and ionomycin) caused aggregation that varied with [Ca] and far exceeded that provoked by Ca alone. Glutaraldehyde-fixed cells did not respond to ionophores with or without Ca. (
iii
) Calcium antagonists inhibited aggregation. These included inhibitors of the Ca-calmodulin complex (
N
-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride and 1-[bis(
p
-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxyl)phenylethyl]imidazolinium chloride), Ca channel blockers (La, Co, Cd, and verapamil), and three nonsteroidal anti-inflammatory agents (indomethacin, ibuprofen, and piroxicam). Results indicated not only that early events of sponge aggregation can be quantified by continuous recording but that aggregation is not simply due to passive agglutination of inert cells by an extracellular proteoglycan. Rather, sponge cells recognize surface ligands to which they respond by Ca-dependent stimulus-response coupling.
Development of translationally active mRNA for larval muscle acetylcholinesterase during ascidian embryogenesis.
Authors: Meedel T H , Whittaker J R
Published: May 2006

Relative quantities of translationally active acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) mRNA present at various developmental stages were compared in embryos of the ascidian Ciona intestinalis. Purified RNA was tested for its translational capacity by microinjection into Xenopus laevis oocytes; the acetylcholinesterase produced was immunoprecipitated with antibody to Ciona acetylcholinesterase and enzyme activity was assayed radiometrically. With this protocol, enzyme synthesis was found to be directly related to the amount of RNA injected and to the oocyte incubation time. A functional template for acetylcholinesterase was first detected at 6 hr of development (late gastrula) and is probably present as early as 5 hr. The level of this template activity increased until the middle tail formation stage (11-12 hr after fertilization) and then remained constant until 16 hr of development (the final stage examined), 2 hr before hatching. These findings, and the results of previous actinomycin D inhibition experiments, indicate that mRNA for ascidian larval muscle acetylcholinesterase is first synthesized during gastrulation.
Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells.
Authors: Shapira G , Stachelek J L , Letsou A , Soodak L K , Liskay R M
Published: May 2006

Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.
Molecular cloning and sequence of partial cDNA for interferon-induced (2'-5')oligo(A) synthetase mRNA from human cells.
Authors: Merlin G , Chebath J , Benech P , Metz R , Revel M
Published: May 2006

By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells. A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes. A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified. In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells. In Namalva cells, mainly one RNA of 1.8 kilobases is seen.
Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments.
Authors: Hatfield D L , Dudock B S , Eden F C
Published: May 2006

A naturally occurring opal suppressor serine tRNA has been purified from chicken liver and used as a probe to isolate the corresponding gene from a library of chicken DNA in bacteriophage lambda. This minor tRNA is encoded by a single-copy gene that is not part of a tRNA gene cluster. DNA sequence analysis of the gene and its flanking DNA segments shows that the gene is encoded in an 87-base-pair segment without intervening sequences and specifies a tRNA that reads the termination codon UGA. This gene has additional nucleotides in the 5' internal promoter region but has a normal 3' internal promoter sequence and the usual termination signal.
Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.
Authors: Herman A , Parham P , Weissman S M , Engelhard V H
Published: May 2006

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.
Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites.
Authors: Barbaccia M L , Gandolfi O , Chuang D M , Costa E
Published: May 2006

Imipramine inhibits the serotonin uptake by binding with high affinity to regulatory sites of this uptake located on axons that release serotonin. The number of imipramine recognition sites located on crude synaptic membrane preparations is reduced by two daily injections of imipramine or desmethylimipramine for 3 weeks. When the binding sites for [3H]imipramine are down-regulated the Vmax of the neuronal uptake of serotonin is increased. Moreover, in minces prepared from the brain hippocampus of rats receiving imipramine in a dose regimen that reduces the number of [3H]imipramine recognition sites, the efficiency of imipramine as a blocker of the serotonin uptake is diminished. Hence the high-affinity binding sites for [3H]imipramine may have a physiological role in modulation of serotonin reuptake. Probably this is mediated by an endogenous effector of these regulatory sites. A nonpeptidic constituent of rat brain capable of displacing [3H]imipramine from its high-affinity binding site and of inhibiting the serotonin uptake in a dose-related manner has been extracted and its partial purification is described.
Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses.
Authors: Merion M , Schlesinger P , Brooks R M , Moehring J M , Moehring T J , Sly W S
Published: May 2006

Like many physiological ligands, several viruses and toxins enter mammalian cells through receptor-mediated endocytosis. Once internalized, the nucleic acids of several viruses and the toxic subunit of diphtheria toxin gain access to the cytosol of the host cell through an acidic intracellular compartment. In this report, we present evidence that one class of mutants of Chinese hamster ovary (CHO)-K1 cells, which is "cross-resistant" to Pseudomonas exotoxin A, diphtheria toxin, and several animal viruses, has a defect in acidification of the endosome. Cells were allowed to internalize fluorescein isothiocyanate-conjugated dextran before subcellular fractionation. Fluorescence measurements on subcellular fractions permitted measurement of the internal pH of the isolated endosomes and lysosomes. Our results show that (i) endosomes and lysosomes from CHO-K1 cells maintain an acidic pH, (ii) acidification of both endosomes and lysosomes is mediated by a Mg2+/ATP-dependent process, (iii) GTP can satisfy the ATP requirement for acidification of lysosomes but not of endosomes, and (iv) at least one class of mutants that is cross-resistant to toxins and animal viruses has a defect in the ATP-dependent acidification of their endosomes. These studies provide biochemical and genetic evidence that the mechanisms of acidification of endosomes and lysosomes are distinct and that a defect in acidification of endosomes is one biochemical basis for cross-resistance to toxins and viruses.
Removal of extraplatelet Na+ eliminates indomethacin-sensitive secretion from human platelets stimulated by epinephrine, ADP, and thrombin.
Authors: Connolly T M , Limbird L E
Published: May 2006

We have previously observed that removal of extraplatelet Na+ markedly diminishes human platelet aggregation and secretion in response to epinephrine. The present studies demonstrate that this effect of the removal of extraplatelet Na+ on platelet function is not unique to activation of platelets by alpha 2-adrenergic agents but represents a phenomenon also evident for other platelet stimuli. Thus, platelet aggregation and secretion in response to maximal concentrations of ADP and lower concentrations of thrombin (less than 0.04 unit/ml) were also markedly reduced in platelets in "Na+-free" medium, suggesting that these agents share an effector mechanism that is similarly inhibited by the removal of extraplatelet Na+. In contrast, platelet aggregation and secretion in response to higher concentrations of thrombin (greater than or equal to 0.04 unit/ml) and to 0.04-1.0 microM (15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), an endoperoxide analog, were identical in control platelets and in those suspended in "Na+-free" medium, indicating that platelets suspended in "Na+-free" medium are functionally intact, at least in response to some stimuli. Furthermore, the observation that U46619 can elicit platelet aggregation and secretion independently of extraplatelet Na+ indicates that the loss of platelet responsiveness to epinephrine, ADP, and low concentrations of thrombin cannot be attributed to a loss of sensitivity to the stimulus-provoked secondary mediator(s) of platelet function, endoperoxides or thromboxane A2. Treatment with indomethacin to block the secondary aggregation and secretion pathways of platelets reduced the aggregatory and secretory responses of control platelets induced by epinephrine, ADP, and low concentrations of thrombin to those characteristic of platelets suspended in "Na+-free" medium. In contrast, indomethacin did not alter the functional responses induced by these agents in platelets suspended in "Na+-free" medium, suggesting that "primary" aggregation is intact but that the "secondary" aggregation and secretion mediated by arachidonic acid metabolites are eliminated by removal of extraplatelet Na+. Consistent with this interpretation is the observation that the indomethacin-insensitive aggregation and secretion induced by U46619 and higher concentrations of thrombin were retained in platelets suspended in "Na+-free" medium. Thus, the responses eliminated by removal of extraplatelet Na+ are those eliminated by treating control platelets with indomethacin, suggesting a strong link between the presence of extraplatelet Na+ and the operation of platelet function mediated by the cyclooxygenase pathway.
DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems.
Authors: Plasterk R H , Brinkman A , van de Putte P
Published: May 2006

The gene product of bacteriophage Mu gin catalyzes a 3,000-base-pair inversion in the DNA of the phage, thus changing its host range. In some strains of Escherichia coli there is a function that can complement Mu gin mutations. This function (pin) was cloned and shown to catalyze an inversion of 1,800 base pairs in the adjacent E. coli DNA (P region). pin- derivatives carry the P region frozen in the (+) or (-) orientation. The function of the switch is not yet clear. The sequences of gin and pin were determined; they exhibit 70% homology. The sequences around the recombination sites of Gin and Pin are also largely homologous; a consensus sequence is derived for the recombination sites of Gin and Pin, and of Hin in Salmonella typhimurium. The amino acid sequences of Gin, Pin, Hin, and TnpR are compared, and the evolutionary relationship between these prokaryotic site-specific recombination systems is discussed.
A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2.
Authors: Weinberg D H , Ketner G
Published: May 2006

Cell lines that produce viral gene products and that can support the growth of viral mutants lacking those products have been valuable in the genetic analysis of the transforming regions of several animal viruses. To extend the advantages of such complementing cell systems to regions of the adenoviral genome not directly involved in transformation, we have constructed a cell line that will support the growth of a defective adenoviral deletion mutant, H2dl808, that lacks most of early region 4 (E4). The right-hand terminal adenovirus 5 EcoRI restriction fragment, which contains all of E4, was first inserted into the vector pSV2gpt, and the recombinant plasmid was introduced into Vero cells by calcium phosphate precipitation. Clones containing the hybrid plasmid were selected by their resistance to mycophenolic acid. Five mycophenolic acid-resistant clones were then tested for the ability to support the growth of H2dl808. One of the five lines, W162, permits plaque formation by H2dl808 at an efficiency that is greater than 10(6)-fold higher than that of the parental Vero cells and allows the production of high-titer, helper-free H2dl808 stocks. Thus, W162 cells are permissive for at least one defective E4 mutant. The line carries, as expected, an intact E4, detected by hybridization. Using an H2dl808 lysate produced on W162 cells, we have accurately mapped the 808 deletion. It extends from between BclI and SmaI sites at positions 91.4 and 92.0, respectively, to just beyond a HindIII site at position 97.2 and, therefore, falls entirely within E4. H2dl808 and W162 should be of value in determining the physiological role of E4 in adenoviral infection.
Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad.
Authors: Glass C , Pittman R C , Weinstein D B , Steinberg D
Published: May 2006

The metabolic fate of homologous high density lipoprotein (HDL) was studied in the rat, tracing the apoprotein A-I (apo A-I) and cholesterol ester moieties simultaneously. The apo A-I was labeled with covalently linked 125I-labeled tyramine cellobiose, which accumulates in the cells degrading the apoprotein; [3H]cholesterol ethers, which cannot be hydrolyzed or mobilized after uptake, were incorporated into the lipid core of reconstituted HDL to reflect the fate of the cholesterol esters. Several lines of evidence, including direct comparison with biologically labeled HDL, are presented to support the validity of this approach. The liver was the major organ of cholesterol ether uptake, accounting for 65% of the total; the adrenal gland and ovary were the most active organs per gram (wet) of weight. Uptake of cholesterol ether was 7-fold greater than that of apo A-I in adrenal, 4-fold greater in the ovary, and greater than 2-fold greater in the liver. The remaining tissues took up apo A-I and cholesterol ethers at more nearly equal rates. Transfer of HDL-associated cholesterol ethers and 125I-labeled apo A-I to other lipoprotein fractions was not observed; thus, the results reflect direct uptake from HDL itself. Whereas uptake of low density lipoprotein appears to involve endocytosis of intact particles, uptake of HDL in at least some rat tissues involves additional, more complex, transfer mechanisms.
Slow cruciform transitions in palindromic DNA.
Authors: Gellert M , O'Dea M H , Mizuuchi K
Published: May 2006

Extrusion of cruciform structures in self-complementary regions of DNA is known to be favored by negative supercoiling of DNA. We show here that, in moderately supercoiled DNA, cruciform extrusion is a very slow process. In plasmid pUC7 DNA, with a 48-base-pair palindrome, the half-time of extrusion at 50 degrees C is typically several hours; rates are even slower at lower temperature. The rates increase significantly with increasing DNA supercoiling but are only slightly faster in DNA species with much longer palindromes. The reabsorption of cruciform arms is also very slow. The equilibrium between cruciform and regular DNA structures is sensitive to changes in the linking number. Measurement of this equilibrium leads to an estimate of 18 kcal/mol (75.3 kJ/mol) for the free energy required to generate a cruciform structure. In bacterial cells, cruciform DNA may be rare, even when it is thermodynamically favored, because of its slow formation.
Isolation of Z-DNA-containing plasmids.
Authors: Thomae R , Beck S , Pohl F M
Published: May 2006

A purified, monoclonal antibody, specific for the left-handed Z-form of poly(dG-dC), was coupled covalently to Sephacryl S-1000 beads. Such an antibody column provides a convenient method to isolate and purify those plasmid DNAs that contain Z-DNA from a large excess of other DNAs, RNA, etc. From a library of Escherichia coli DNA, cloned into the vector plasmid pUC-8, several recombinant plasmids were isolated, which bind to this antibody. Thus, E. coli contains sequences, which in "natural" negatively supercoiled DNA, adopt a left-handed Z-DNA-like conformation.
Thermotaxis of
Dictyostelium discoideum
amoebae and its possible role in pseudoplasmodial thermotaxis
Authors: Hong Choo B. , Fontana Donna R. , Poff Kenneth L.
Published: May 2006

Thermotaxis by individual amoebae of
Dictyostelium discoideum
on a temperature gradient is described. These amoebae show positive thermotaxis at temperatures between 14°C and 28°C shortly (3 hr) after food depletion. Increasing time on the gradient reduces the positive thermotactic response at the lower temperature gradients (midpoint temperatures of 14, 16, and 18°C), and amoebae show an apparent negative thermotactic response after 12 hr on the gradient. The thermotaxis response curve for “wild-type” amoebae after 16 hr on the gradient is similar to that shown for the pseudoplasmodia. Growth of the amoebae at a different temperature causes a shift in the thermotaxis response curve for the amoebae. This adaptation is similar to that shown for the pseudoplasmodia. Two mutants in thermotaxis, HO428 and HO813, show changes in amoebal thermotaxis similar to the observed changes in pseudoplasmodial thermotaxis. On the basis of the similarities between these responses, thermotaxis by the amoebae is proposed to be the basis for thermotaxis by the multicellular pseudoplasmodium.
Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2.
Authors: Jariwalla R J , Aurelian L , Ts'o P O
Published: May 2006

Diploid Syrian hamster embryo (SHE) cells were passaged after transfection with recombinant plasmids containing herpes simplex virus type 2 (HSV-2) DNA inserts Bgl II focus-forming fragment N, Bgl II transforming fragment C, and EcoRI/HindIII fragment AE. Cultures transfected with salmon DNA or with 0.1-5.0 micrograms of Bgl II fragment N reached crisis and senesced. Those transfected with 0.1-0.5 micrograms of Bgl II fragment C or its left-hand 64% subclone EcoRI/HindIII fragment AE escaped senescence and formed continuous lines. At early passages, these lines as well as isolated clones grew in 2% serum but formed small (less than or equal to 0.1 mm) colonies in 0.3% agarose and were nontumorigenic. Serial passaging of Bgl II fragment C-induced cultures and isolated clones resulted in the appearance of large (greater than 0.25 mm) colonies in agarose followed by tumorigenicity. This behavior was not exhibited by the EcoRI/HindIII fragment AE-induced cultures that remained nontumorigenic after 53 passages. DNA from normal SHE cells exhibited homology to Bgl II fragment C but, under relatively stringent conditions, DNAs from transformed and tumor-derived lines exhibited discrete hybridizing bands comigrating with authentic viral fragments. These results indicate that neoplastic transformation of normal diploid SHE cells by HSV-2 DNA fragments involves at least two distinct steps--i.e., immortalization and conversion to tumorigenicity. EcoRI/HindIII fragment AE representing the left 64% of Bgl II fragment C is sufficient to induce immortalization. However, DNA sequences from both left-hand 64% and right-hand 36% subfragments of Bgl II fragment C are required for tumorigenic transformation.
Induction of T-cell maturation by a cloned line of thymic epithelium (TEPI).
Authors: Beardsley T R , Pierschbacher M , Wetzel G D , Hays E F
Published: May 2006

A cloned cell line of thymic origin has been characterized as epithelial in nature. A description of the procedures for derivation and cloning of the cell line includes use of epidermal growth factor. The thymic epithelial (TEPI) cell line is Ia antigen positive, forms desmosomes, and produces an extracellular fibronectin matrix. The supernatant from confluent monolayers of TEPI was tested for its ability to promote thymocyte functional activity. TEPI supernatant (TEPI SN) was demonstrated to greatly enhance the response of peanut agglutinin-positive thymocytes to alloantigen, as measured by cell-mediated lympholysis. Furthermore, preincubation of peanut agglutinin-positive thymocytes with TEPI SN prior to allostimulation resulted in marked enhancement, thus distinguishing it from interleukin 2. Finally, TEPI SN was demonstrated to induce interleukin 2 production by peanut agglutinin-positive thymocytes in the presence of concanavalin A. This activity was demonstrated not to be due to interleukin 1, which is absent in TEPI SN. Preliminary biochemical analysis indicates that the biological activity is associated with a Mr 50,000 entity. The data suggest that TEPI produces a soluble factor capable of inducing function of an immature thymocyte subpopulation into an IL 2 producer.
Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and Ia molecules.
Authors: Matis L A , Glimcher L H , Paul W E , Schwartz R H
Published: May 2006

The activation of specific T-cell clones by antigens is dependent upon the corecognition of restriction elements expressed on antigen-presenting cells (APC). For clones that respond by proliferation and lymphokine production, the restriction element is usually an Ia (I-region-associated) molecule. We show here that the magnitude of the proliferative response of such clones is a function of the product of antigen concentration and the number of Ia molecules expressed on APC. This conclusion was reached through the study of antigen concentration-response curves of T-cell clones specific for pigeon cytochrome c. These curves are characterized by a peak in thymidine incorporation, followed by a decrease in the magnitude of the response as antigen concentrations are increased. The decline in response at high concentrations was not the consequence of the emergence of suppressor cells in the APC population, as it was observed when cells of a cloned B-cell hybridoma line were used to present pigeon cytochrome c to these T-cell clones. The critical role of both antigen concentration and the number of Ia molecules on the APC in determining the magnitude of proliferation was demonstrated in several ways. (i) An inverse relationship was observed between the antigen concentration required for maximum proliferation and the number of APC present in culture. At high antigen concentrations, which caused responses less than maximum, reducing the number of APC actually increased the magnitude of the antigen-induced proliferation. (ii) Decreasing the number of relevant Ia molecules per APC (i.e., as in F1 hybrids) resulted in the requirement for an increased antigen concentration for maximal response and in enhanced proliferation at high antigen concentrations. (iii) In the presence of anti-Ia antibody, higher concentrations of antigen were required for maximal response; at high antigen concentrations, proliferation was enhanced in the presence of a monoclonal anti-Ia antibody directed against the Ia restriction element.
Induction of immune responses in vivo with small numbers of veiled (dendritic) cells.
Authors: Knight S C , Mertin J , Stackpoole A , Clark J
Published: May 2006

The role of dendritic or veiled cells (VC) from lymph nodes or spleens of rats and mice in initiating immune responses in vivo has been investigated. Host-versus-graft responses were induced by injection of VC from spleens of (C57BL/10 X CBA) F1 mice into the footpads of parental strain (CBA) animals and measured by the increase in the weight of the draining popliteal lymph nodes. The potency of VC to induce the responses was 100-fold greater than that of unseparated spleen cells. The in vivo effect of VC was not limited to this direct allogeneic stimulation because autologous VC could also be used in the induction of an experimental autoimmune disease. In these studies, experimental allergic encephalomyelitis was produced in Lewis rats by injection of guinea pig brain and spinal cord material emulsified in Freund's complete adjuvant. Small numbers of VC from spleens or lymph nodes of rats showing clinical signs of experimental allergic encephalomyelitis induced a mild form of the disease when injected intravenously into normal Lewis rats. Thus, VC carrying antigen, either as an integral part of their surface membrane or acquired during exposure to antigenic substances, appeared to be very potent agents for the induction of immune responses.
Offspring sex ratio in red-winged blackbirds is dependent on maternal age
Authors: Blank James L. , Nolan Val
Published: May 2006

In a marsh-breeding population of red-winged blackbirds, the sex ratio of offspring that survived to leave the nest varied with maternal age. Old mothers produced an excess of male fledglings, middle-aged mothers produced almost equal proportions of males and females, and young mothers produced nearly twice as many females as males. More males than females hatched from the eggs of old mothers, whereas among newly hatched progeny of middle-aged and young mothers the sex ratio did not differ from unity. The hatching rate of eggs of old mothers was unusually low, suggesting that the biased sex ratio of their hatchlings may have been caused by more frequent death of female embryos, although other possibilities can be imagined. Starvation of nestlings after hatching also affected the sex ratio among young that left the nest. When starvation occurred, it fell principally on young produced by the last and next-to-last eggs laid in the clutch. Because old mothers allocated relatively more energy to those eggs than to earlier-laid eggs, whereas young mothers apportioned energy equally to their eggs, few nestlings of old mothers but many nestlings of young mothers starved. Most nestlings that died were male. It followed that the male bias in sex ratio of progeny of old mothers did not change between hatching and nestleaving, but the ratio among progeny of young mothers shifted after hatching to a strong bias favoring females at nest-leaving.
Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.
Authors: Edlund T , Ny T , Rånby M , Hedén L O , Palm G , Holmgren E , Josephson S
Published: May 2006

We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.
Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors
Authors: Carraway Coralie A. Carothers , Jung Goeh , Carraway Kermit L.
Published: May 2006

The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-C1) and mobile (MAT-B1) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-C1 cells with Triton X-100-containing buffers gave insoluble residues showing three major components by NaDodSO
4
/polyacrylamide gel electrophoresis: actin, a 58,000-dalton polypeptide, and a cell surface glycoprotein of 75,000-80,000 daltons. The ratio of these components in Triton X-100-insoluble residues, as determined by scintillation counting of bands from gels of [
3
H]leucine-labeled microvillar membranes, approached equimolar, suggesting a specific complex of the components. The three components of the putative complex cosedimented on sucrose density gradients of Triton X-100/buffer-treated membranes. Gel filtration on Sepharose 2B gave a peak included in the column that contained only the glycoprotein, actin, and 58,000-dalton polypeptide by one-dimensional NaDodSO
4
electrophoresis and by two-dimensional isoelectric focusing/NaDodSO
4
electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-B1 microvillar membranes by Sepharose 2B gel filtration in Triton X-100-containing buffers contained actin and the glycoprotein but no 58,000-dalton polypeptide. From these results we propose that the cell surface-cytoskeleton interactions in the 13762 tumor cell microvilli involve direct association of actin with the cell surface glycoprotein. We further suggest that the 58,000-dalton polypeptide stabilizes the association of this complex with the microfilaments in the MAT-C1 microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.