bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the interleukin 2 receptor beta chain using three distinct monoclonal antibodies.
Authors: Tsudo M , Kitamura F , Miyasaka M
Published: May 2006

The human high-affinity receptor for interleukin 2 (IL-2) has been proposed as being a membrane complex composed of at least two distinct polypeptide chains: p55 (alpha chain), recognized by the anti-Tac monoclonal antibody (mAb), and p75 (beta chain), both of which are capable of binding IL-2. Whereas the alpha chain itself has been shown to be nonfunctional, the beta chain appears to be pivotal in the IL-2 signal transduction, although the beta chain is otherwise poorly characterized. Three beta chain-specific mAbs, designated Mik-beta 1, -beta 2, and -beta 3, were developed. Mik-beta 1 and -beta 2 completely inhibited the IL-2 binding to the beta chain, whereas Mik-beta 3 immunoprecipitated the beta chain crosslinked with 125I-labeled IL-2. The beta chain immunoprecipitated by these mAbs was revealed to have a Mr of 68,000-72,000. High-affinity IL-2 binding was completely abolished by Mik-beta 1. Although IL-2-dependent T-cell growth at high IL-2 concentrations was not inhibited by the anti-Tac, it was almost completely inhibited by Mik-beta 1 in the presence of the anti-Tac. These results clearly indicate that the beta chain is an indispensable component to the high-affinity IL-2 receptor and is responsible for the IL-2 signal transduction. The beta chain was found to be constitutively expressed without the alpha chain on the surface of peripheral blood Leu-19+ natural killer cells.
High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid.
Authors: Steck T R , Close T J , Kado C I
Published: May 2006

To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.
In vitro transcription directed from the somatostatin promoter is dependent upon a purified 43-kDa DNA-binding protein.
Authors: Andrisani O M , Zhu Z N , Pot D A , Dixon J E
Published: May 2006

In vitro transcription analyses were used to establish the biological function of a 43-kDa affinity-purified DNA-binding protein. The 43-kDa affinity-purified protein protects the region from position -59 to position -35 of the somatostatin promoter from DNase I digestion. This region of the somatostatin promoter harbors the TGACGTCA motif, also found and required for function in a number of other cAMP-responsive and adenovirus E1A-inducible promoters. Efficient and authentic transcription in vitro directed from the somatostatin promoter requires the TGACGTCA promoter element. In vitro transcription assays performed in the presence of somatostatin (positions -60 to -29), enkephalin (positions -105 to -71), and adenovirus type 5 E3 gene (positions -72 to -42) competitor fragments, harboring similar TGACGTCA motifs, selectively inhibit transcription directed from the somatostatin promoter, suggesting that the TGACGTCA element is the site of interaction of a somatostatin gene transactivator. Furthermore, extracts depleted of the TGACGTCA-binding activities by affinity chromatography utilizing a biotinylated oligonucleotide-avidin resin, are incapable of directing transcription from the somatostatin but not from the adenovirus major late promoter. Addition of the purified 43-kDa protein to the affinity-depleted extract restores transcription from the somatostatin promoter. These results are consistent with the 43-kDa protein being a trans-activator of the somatostatin gene.
Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products.
Authors: Crute J J , Tsurumi T , Zhu L A , Weller S K , Olivo P D , Challberg M D , Mocarski E S , Lehman I R
Published: May 2006

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.
Polycythemia in transgenic mice expressing the human erythropoietin gene.
Authors: Semenza G L , Traystman M D , Gearhart J D , Antonarakis S E
Published: May 2006

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5' flanking sequence and 0.7 kilobase of 3' flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.
Major histocompatibility complex class I-restricted activation of cloned T cells by a soluble protein in the absence of accessory cells.
Authors: Dick T , Reske-Kunz A B
Published: May 2006

A T-cell clone, 10BK.1, was established from the draining lymph nodes of (B10 x B10.BR)F1 mice immunized with ovalbumin (OVA) according to standard protocols. Upon coculture with the antigen, 10BK.1 cells reacted by production of lymphokines and by proliferation despite the absence of additional antigen-presenting cells. These T cells do not express major histocompatibility complex (MHC) class II molecules on the cell surface as assessed on the basis of several criteria: by cytofluorometric analysis I-A and I-E determinants were not detectable; 10BK.1 cells could not act as antigen-presenting cells for long-term-cultured MHC class II-restricted T-cell clones; and monoclonal antibodies directed at both MHC class II isotypic complexes (I-A, I-E) did not suppress their OVA-induced activation. In contrast, proliferation of 10BK.1 T cells in response to OVA was abrogated by antibodies directed at H-2Kb antigens. Inhibition experiments employing antibodies directed at Lyt-2 and L3T4 antigens in addition to cytofluorometric analysis revealed that T-cell clone 10BK.1 exhibits the Thy-1+,Lyt-2+,Ly-1-,L3T4- phenotype. 10BK.1 cells pulsed with OVA and fixed with glutaraldehyde induced proliferation of untreated 10BK.1 cells. These data support the theory that 10BK.1 T cells present the exogenous globular protein OVA to one another in an MHC class I-restricted manner, resulting in cell activation and proliferation independent of added accessory cells.
Molecular basis for expression of the A48 regulatory idiotope on antibodies encoded by immunoglobulin variable-region genes from various families.
Authors: Zaghouani H , Bonilla F A , Meek K , Bona C
Published: May 2006

The idiotype defined by the levan-specific BALB/c myeloma protein ABPC48 (A48) has previously been encountered only in antibodies the variable regions of which derive from the VHX24 and V kappa 10 gene families. We have demonstrated expression of the idiotope recognized by the monoclonal anti-A48 idiotype antibody IDA10 on five monoclonal antibodies from different mouse strains, with different specificities including foreign and self antigens and deriving their variable regions from families other than VHX24 and V kappa 10. We analyzed variable region protein structure (deduced from nucleotide sequences) and hydrophilicity profiles of idiotype+ and idiotype- antibodies. We identified four surface-exposed areas (one in the heavy chain and three in the light chain) that may contribute to expression of the idiotope defined by antibody IDA10.
Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro.
Authors: Biggs B A , Kemp D J , Brown G V
Published: May 2006

Subtelomeric deletions are responsible for the loss of expression of several Plasmodium falciparum antigens, including the knob-associated histidine-rich protein (KAHRP). Such deletions are detectable by two-dimensional pulsed-field gradient electrophoresis (PFGE) in which the chromosomes separated in dimension 1 are cleaved with Apa I, and the sizes of telomeric fragments are determined in dimension 2. This sensitive technique has enabled us to examine the role of subtelomeric deletions in two aspects of the biology of Plasmodium falciparum. First, we show that similar subtelomeric deletions to those that occur in vitro also occur in field isolates. Second, we demonstrate a correlation between subtelomeric deletions and loss of the phenotype of "cytoadherence" in cultured isolates. Subclones were generated from the cytoadherent cloned isolate ItG2F6, and their phenotypes were examined with respect to cytoadherence, the expression of "knobs," and agglutination of infected erythrocytes with rabbit antiserum. The only chromosomal change detectable by two-dimensional PFGE among subclones that differ from wild type in each of these three characteristics is a deletion of approximately 100 kilobases at one end of chromosome 2. This deletion includes the gene coding for KAHRP and the subtelomeric repeat designated rep20.
Prostaglandin E2 and its methyl ester reduce cataplexy in canine narcolepsy.
Authors: Nishino S , Mignot E , Fruhstorfer B , Dement W C , Hayaishi O
Published: May 2006

The effects of intravenous administration of prostaglandins (PGs) were investigated in genetically narcoleptic Doberman pinschers. The treatment of narcoleptic dogs with PGE2 and PGE2 methyl ester, but not PGD2 and PGD2 methyl ester, induced a dose-dependent reduction of canine cataplexy, a dissociated manifestation of rapid-eye-movement sleep. The effect was specific and not associated with any change in other behavior. Furthermore, the effect was long-lasting (up to 2 hr) and could not be explained by the acute cardiovascular changes seen after intravenous PG administration. PGE2 methyl ester, a lipophilic derivative of PGE2 with more central penetration than PGE2, was 4 times more potent than PGE2. These results indicate that PGE2 modifies cataplexy through a central effect and suggest that this prostaglandin may play a role in rapid-eye-movement sleep regulation.
Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices.
Authors: Stratton K R , Worley P F , Huganir R L , Baraban J M
Published: May 2006

We have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.
Expression of the U1 RNA gene repeat during early sea urchin development: evidence for a switch in U1 RNA genes during development.
Authors: Santiago C , Marzluff W F
Published: May 2006

The majority of the genes for U1 RNA are organized in tandemly repeated units in the sea urchin. To assess the level of expression of these genes in the sea urchin Lytechinus variegatus, we measured the transcription of sequences 3' to the gene. The tandemly repeated U1 genes are expressed in morula and continue to be expressed at high rates until 2 hr after hatching, at which time the rate of expression of all the U1 genes and the tandemly repeated U1 genes declines sharply. By the gastrula stage the synthesis of total U1 RNA has declined by a factor of 8. The major tandemly repeated genes are inactive by this time, although other U1 genes remain active. The sequence of U1 RNA synthesized late in embryonic development differs from the sequence of U1 RNA encoded by the tandemly repeated set of U1 RNA genes, indicating that there must be other U1 RNA genes that are active late in embryonic development.
Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence.
Authors: Relman D A , Domenighini M , Tuomanen E , Rappuoli R , Falkow S
Published: May 2006

Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.
Activation of glucose uptake by insulin and insulin-like growth factor I in Xenopus oocytes.
Authors: Janicot M , Lane M D
Published: May 2006

Xenopus laevis oocytes possess a glucose transport system that is activated 3- to 5-fold by insulin-like growth factor I (Ka = 3 nM) and insulin (Ka = 200-250 nM), properties suggesting activation mediated by an insulin-like growth factor I receptor. This activation increases the Vmax of hexose uptake and has little or no effect on the Km for deoxyglucose (Km = 1-2 mM). Activation by hormone requires about 60 min and is inhibited by cytochalasin B but not by cycloheximide. The dependence of hexose uptake rate on hexose concentration exhibits cooperativity with Hill coefficients of 1.8 and 1.4 for the basal and hormone-activated states, respectively. Microinjection of a monoclonal antibody directed against the tyrosine kinase domain of the human insulin receptor blocks activation of hexose uptake by insulin-like growth factor I and insulin but has no effect on basal uptake. Taken together the results implicate the tyrosine-specific protein kinase activity of a cell-surface insulin-like growth factor I receptor in the activation of glucose transport in the Xenopus oocyte.
Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions.
Authors: Sáez J C , Connor J A , Spray D C , Bennett M V
Published: May 2006

Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca2+, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca2+ concentration, [Ca2+]i, is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP3), a second messenger that can evoke Ca2+ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca2+ and InsP3 in freshly isolated pairs or small clusters of rat hepatocytes. The Ca2+ indicator fura-2 was used to monitor transjunctional diffusion of Ca2+ directly or to detect passage of InsP3 by localized Ca2+ release. Fura-2 injected as the free acid passed between cells. Injection of InsP3 or CaCl2 immediately increased [Ca2+]i in the injected cell (peak values less than 1 microM), and [Ca2+]i increased rapidly in contacting cells (within seconds). The initial rise in [Ca2+]i induced by InsP3 was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca2+. In the coupled cells the regions of greatest increase were not necessarily near the contact zone. In contrast, the rise induced in [Ca2+]i by CaCl2 injection when cells were bathed in normal Ca2+ was always more diffuse than with InsP3 injection, and in cells coupled to a cell injected with CaCl2 the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP3 (or an active metabolite, but not Ca2+) diffused between cells to cause localized release of Ca2+ from intracellular stores. Ca2+ injection induced a rise in [Ca2+]i in coupled cells even when cells were maintained in Ca2+-free saline, suggesting that changes in [Ca2+]i seen in adjacent cells were due to transjunctional diffusion from the injected cell and not to uptake from the extracellular solution. However, in Ca2+-free saline, [Ca2+]i distribution was nonuniform, indicating that Ca2+-releasing mechanisms contribute to the observed changes. No increase in [Ca2+]i was seen in adjacent cells when Ca2+ was injected after treatment with the uncoupling agent octanol (500 microM), which itself did not change [Ca2+]i. These data provide evidence that the second messengers Ca2+ and InsP3 can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.
Direct visualization of the interrelationship between intramembrane and extracellular structures.
Authors: Wade J B , Coleman R A
Published: May 2006

The apical surface of the toad urinary bladder is covered by an interconnected mesh of glycocalyx, which appears to attach to the plasma membrane bilayer. To evaluate the interrelationship between these extracellular elements and intramembrane structures, a strategy was devised to produce composite replicas that allow the simultaneous visualization of intramembrane particles by freeze-fracture while the glycocalyx mesh is replicated by rotary shadowing of the extracellular surface after freeze-drying. Evaluation of these composite replicas by electron microscopy reveals that contacts occur between extracellular filamentous elements and intramembrane particles. This structural organization may be important for stabilizing intramembrane components and for anchoring extracellular elements to the membrane.
Effects of recombinant insulin-like growth factor I on insulin secretion and renal function in normal human subjects.
Authors: Guler H P , Schmid C , Zapf J , Froesch E R
Published: May 2006

Insulin-like growth factor I (IGF-I) is an important mediator of growth hormone (GH) action and it appeared tempting to evaluate possible clinical applications. Recombinant IGF-I was infused s.c. at a dose of 20 micrograms/kg of body weight per hour during 6 days in two healthy adult subjects. Blood glucose and fasting insulin levels remained within normal limits and IGF-II levels were suppressed. In contrast to insulin, fasting C peptide levels were decreased. GH secretion was also suppressed by IGF-I. Our preliminary data allow us to distinguish between the effects of GH per se and those of IGF-I: GH causes hyperinsulinism, whereas IGF-I leads to decreased insulin secretion. Glomerular filtration rate, as estimated by creatinine clearance, increased to 130% of preinfusion values during the IGF-I infusion. Total creatinine and urea excretion remained unchanged. We conclude that IGF-I influences kidney function and, in contrast to GH, exerts an insulin-sparing effect. It may be speculated that the therapeutic spectrum of IGF-I is quite different from that of GH.
Ontogenesis and localization of Ca2+ channels in mammalian skeletal muscle in culture and role in excitation-contraction coupling.
Authors: Romey G , Garcia L , Dimitriadou V , Pincon-Raymond M , Rieger F , Lazdunski M
Published: May 2006

The mechanism of excitation-contraction (E-C) coupling in skeletal muscle is not yet well established. Cultured mouse skeletal muscle cells have been used to study the relationships between triad formation, Ca2+ channel activities, and contractions. The ontogenesis of voltage-dependent Ca2+ channels and their localization in relation to the ability of muscle to contract and the ultrastructural organization of sarcomeres and triads have been investigated by using an electrophysiological approach together with an electron microscope study. At an early stage of development, both fast (Ifast) and slow (Islow) types of Ca2+ channels are found at the surface membrane. At later stages of development, fast Ca2+ channels remain at the surface membrane, while slow Ca2+ channels migrate to the transverse-tubule membrane. The voltage dependence of fast Ca2+ channels compared to the voltage dependence of contraction clearly shows that these Ca2+ channels have no direct role in E-C coupling. Detubulation at all stages of development has confirmed that T tubules contain essential elements for E-C coupling. However, this work also shows that Ca2+ flowing through slow Ca2+ channels situated in the T-tubular system is not important for contraction. Myotubes lacking slow Ca2+ channels or having no slow Ca2+ channel transport activity (jumps to high membrane potentials, no external Ca2+, block of Islow by Co2+) still retain contraction.
Wall-thickness and midwall-radius variations in ventricular mechanics.
Authors: Chadwick R S , Ohayon J , Lewkowicz M
Published: May 2006

A fluid-fiber-collagen stress tensor is used to describe the rheology of the left ventricle of the heart. Linear theory is used to find the equilibrium solutions for the end-diastolic and end-systolic states of general axisymmetric shapes that are small perturbations of a thick-walled finite cylinder. The general problem can be studied by superposing the effects of variable midwall radius but constant wall thickness with those of variable wall thickness but constant midwall radius. A Fourier series representation is used to describe the midwall radius and thickness functions. Numerical calculations are performed to determine the deformed geometry and spatial distributions of tissue pressure, stresses, and fiber strains. The calculations proved to be highly accurate when compared to an analytical solution obtained for the special case of no fibers. The results show significant longitudinal differences when compared to results for the cylindrical geometry, with more sensitivity to variation in wall thickness than to variation in midwall radius.
Heterologous desensitization of bombesin-induced mitogenesis by prolonged exposure to vasopressin: a post-receptor signal transduction block.
Authors: Millar J B , Rozengurt E
Published: May 2006

Prolonged exposure of quiescent Swiss 3T3 cells to vasopressin prevents mitogenic stimulation on subsequent addition of bombesin. This heterologous desensitization is selective and can be mimicked by vasopressin agonists, including [Lys8]vasopressin and oxytocin but not by the V1-type-specific vasopressin receptor antagonist [Pmp1,O-Me-Tyr2,Arg8]vasopressin [where Pmp is 1-(beta-mercapto-beta,beta-cyclopenthamethylene propionic acid)]. Furthermore, vasopressin-induced loss of responsiveness to bombesin can be blocked by addition of this antagonist, indicating that heterologous desensitization is mediated through the vasopressin receptor. Desensitization requires prolonged incubation (half-maximal desensitization occurring after approximately 20 hr of pretreatment) and continuous protein synthesis. Bombesin responsiveness is restored by incubation in the absence of vasopressin. Pretreatment does not alter the number, affinity, or internalization capacity of the bombesin receptors. However, the induction of the protooncogene c-fos by bombesin is profoundly inhibited after vasopressin pretreatment. We suggest that the coupling of the activated bombesin receptor to the generation of its early signals is impaired in desensitized cells.
Wheat tetrameric inhibitors of insect α-amylases: Alloploid heterosis at the molecular level
Authors: Gomez Luis , Sanchez-Monge Rosa , Garcia-Olmedo Francisco , Salcedo Gabriel
Published: May 2006

Tetrameric inhibitors of heterologous α-amylases have been characterized in allohexaploid wheat,
Triticum aestivum
(genomes AABBDD), as well as in
Triticum turgidum
(AABB) and
Triticum tauschii
(DD). Their subunits have been identified as the previously described CM proteins. Single oligomeric species were observed in
T. Turgidum
(subunits CM2, CM3A, and CM16) and in
T. tauschii
(CM1, CM3D, and CM17) by a two-dimensional electrophoretic method that does not dissociate the inhibitors in the first dimension. Multiple tetrameric species, resulting from different combinations of the subunits contributed by the two ancestral species, are observed by the same procedure in
T. aestivum.
The three types of subunits were required for significant activity when the inhibitor of
T. turgidum
was reconstituted from the purified subunits, whereas, in the case of
T. tauschii
, binary mixtures involving subunit CM1 also had some activity. Additional combinations of the subunits present in these two species, which occur in the allohexaploid
T. aestivum
, were also reconstituted, and their inhibitory activities ranged from 144% to 33% the activity of the reconstituted inhibitor from
T. tauschii
. The activity of these inhibitors toward the α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) of the insect
Tenebrio molitor
is much greater than that against the salivary enzyme. These observations, together with the previously established chromosomal locations of genes encoding CM proteins, fit a model of alloploid heterosis at the molecular level.
Expression of functional pituitary somatostatin receptors in Xenopus oocytes.
Authors: White M M , Reisine T
Published: May 2006

Functional somatostatin (SRIF, somatotropin release-inhibiting factor) receptors were expressed in Xenopus oocytes after injection of RNA isolated from the anterior pituitary tumor cell line AtT20. SRIF receptors were detected by measuring the ability of SRIF to inhibit cAMP formation stimulated by beta 2-adrenergic agonists in individual oocytes. beta 2-Adrenergic receptors (beta 2ARs) were expressed in oocytes by coinjecting RNA prepared by in vitro transcription of a beta 2AR cDNA clone with the pituitary cell RNA. Uninjected oocytes do not express detectable levels of either beta 2ARs or SRIF receptors. In oocytes coinjected with AtT20 and beta 2AR RNA, on the other hand, isoproterenol treatment led to a 2- to 3-fold increase in cAMP levels, whereas cotreatment with SRIF reduced this accumulation by 50-60%. The SRIF precursor somatostatin-28 and the cyclohexapeptide agonist MK678 also inhibited cAMP formation, whereas the biologically inactive N-terminal 14-amino acid fragment of somatostatin-28 was ineffective. The ability to detect changes in cAMP levels in individual oocytes may provide a simple procedure for the expression cloning of SRIF receptor cDNAs and other receptors functionally coupled to stimulation or inhibition of adenylate cyclase.
Expression of a cloned streptavidin gene in Escherichia coli.
Authors: Sano T , Cantor C R
Published: May 2006

We describe the construction of systems for expressing the cloned streptavidin gene in Escherichia coli. Although the streptavidin gene is extremely lethal to the host cells, because of the strong biotin binding of the gene product, the gene was expressed efficiently in E. coli by using T7 RNA polymerase/T7 promoter expression systems. The expressed streptavidin accumulated to more than 35% of the total cell protein. The expressed streptavidin was insoluble in the cell. However, after solubilization by dialysis against 6 M guanidine hydrochloride (pH 1.5) and removal of guanidine hydrochloride by dialysis, the protein became soluble and renatured. This simple procedure yielded streptavidin purified almost to homogeneity. The purified streptavidin bound 3.5-3.9 molecules of biotin per molecule, indicating that it had almost full biotin-binding ability. Some of the purified streptavidin molecules aggregated into oligomers, suggesting that the C-terminal region of the molecule, present in our material but absent in typical preparations, may be responsible for the aggregation.
Determination of the crystal structure and composition of Li6Be4OH12 by the stochastic method.
Authors: Pauling L
Published: May 2006

Because of the failure to find a structure for LiBeH3 with a face-centered unit cube with edge 5.09 A, the x-ray powder pattern has been reindexed for a body-centered unit cube with edge 7.24 A. Application of the principles of structural chemistry leads to the formula Li6Be4OH12 and to a structure involving Be4OH12 clusters formed by 4 BeOH3 tetrahedra with their O corner shared, Be--(H,O) bond length 1.59 A, and with the clusters joined to one another by Li with octahedral or rectangular-planar coordination of 6 H or 4 H, Li-H bond lengths about 1.92 A.
Progastrin expression in mammalian pancreas.
Authors: Bardram L , Hilsted L , Rehfeld J F
Published: May 2006

Expression and processing of progastrin were examined in fetal, neonatal, and adult pancreatic tissue from five mammalian species (cat, dog, man, pig, and rat). A library of sensitive, sequence-specific immunoassays for progastrin and its products was used to monitor extractions and chromatography before and after cleavage with processing-like enzymes. The results showed that progastrin and its products are expressed in the pancreas of all species in total concentrations varying from 0.3 to 58.9 pmol/g of tissue (medians). The degree of processing was age- and species-dependent. In comparison with adult pancreatic tissue the fetal or neonatal pancreas processed a higher fraction to bioactive, C-terminally amidated gastrin. Nevertheless, the pancreatic processing was always less complete than that of the adult antral mucosa. The moderate level of expression and the attenuated processing in the adult pancreas contribute to explain previous failures to detect gastrin in normal pancreatic tissue. Our results indicate that gastrin-producing tumors in the pancreas are not ectopic, but arise from cells that normally express the gastrin gene.
Induction of transcription from the long terminal repeat of Moloney murine sarcoma provirus by UV-irradiation, x-irradiation, and phorbol ester.
Authors: Lin C S , Goldthwait D A , Samols D
Published: May 2006

The long terminal repeat (LTR) of Moloney murine sarcoma virus (Mo-MuSV) was used as a model system to study the stress response of mammalian cells to physical carcinogens. The chloramphenicol acetyltransferase (CAT) gene was inserted between two Mo-MuSV LTRs, and the LTR-CAT-LTR construct was used for virus production and was integrated into the genome of NIH 3T3 cells in the proviral form. This construct was used to assure that the integrated CAT gene was driven by the promoter of the LTR. Expression of the CAT gene was stimulated 4-fold by UV irradiation, and the peak of activity was observed at 18 hr. In contrast, stimulation of the CAT expression after x-irradiation was 2-fold and occurred at 6 hr. Phorbol myristate acetate also stimulated CAT activity 4-fold with a peak at 6 hr. Down-regulation of protein kinase C blocked totally the response to x-irradiation but only partially the response to UV. The protein kinase inhibitor H7 blocked the response to treatment by UV, x-ray, and phorbol ester.
Thyroid hormone regulates expression of a transfected human alpha-myosin heavy-chain fusion gene in fetal rat heart cells.
Authors: Tsika R W , Bahl J J , Leinwand L A , Morkin E
Published: May 2006

The rat alpha-myosin heavy-chain (alpha-MHC) gene is regulated by 3,5,3'-triiodo-L-thyronine (T3) in ventricular myocardium and is constitutively expressed in atrial tissue. Less is known about regulation of the human gene, but conservation of sequences in the 5'-flanking region between the rat and human alpha-MHC genes suggests that the human gene may be regulated similarly. Accordingly, T3-responsiveness and tissue-specific expression of human and rat alpha-MHC/chloramphenicol acetyltransferase fusion constructs have been compared in rat fetal heart cells, L6E9 myoblasts and myotubes, 3T3 fibroblasts, and HeLa cells. Transient transfection assays revealed a complex series of cis-regulatory elements in the 5'-flanking sequences in the human genes, including a basal promoter element with canonical TATAA and CAAT sequences, two positive regulatory element(s), and two negative regulatory elements, which markedly diminished both constitutive and T3-inducible activity. Interestingly, the human gene seemed to contain a proximal thyroid-hormone response element(s) not found in the rat gene. In L6E9 myoblasts and myotubes, the human constructs were constitutively expressed but not T3-regulated; none of the constructs were active in 3T3 or HeLa cells. We propose that interactions among the thyroid hormone responsive elements and other cis-acting elements in the human alpha-MHC 5'-flanking sequences may be sufficient to explain the characteristic features of expression of this gene in cardiac tissues.
Genomic RNA of an insect virus directs synthesis of infectious virions in plants.
Authors: Selling B H , Allison R F , Kaesberg P
Published: May 2006

Newly synthesized virions of flock house virus (FHV), an insect nodavirus, were detected in plant cells inoculated with FHV RNA. FHV was found in whole plants of barley (Hordeum vulgare), cowpea (Vigna sinensis), chenopodium (Chenopodium hybridum), tobacco (Nicotiana tabacum), and Nicotiana benthamiana and in protoplasts derived from barley leaves. Virions produced in plants contained newly synthesized RNA as well as newly synthesized capsid protein. These results show that the intracellular environment in these plants is suitable for synthesis of a virus normally indigenous only to insects. Such synthesis involves, minimally, translation of viral RNA, RNA replication, and virion assembly. Inoculation of barley protoplasts with FHV virions resulted in synthesis of small amounts of progeny virions, suggesting that FHV virions are capable of releasing their RNA in plant cells. In N. benthamiana, virions resulting from inoculation with RNA were detected not only in inoculated leaves but also in other leaves of inoculated plants, suggesting that virions could move in this plant species. Such movement probably occurs by a passive transport through the vascular system rather than by an active transport involving mechanisms that have evolved for plant viruses.
Relation of spontaneous transformation in cell culture to adaptive growth and clonal heterogeneity.
Authors: Rubin A L , Yao A , Rubin H
Published: May 2006

Cell transformation in culture is marked by the appearance of morphologically altered cells that continue to multiply to form discrete foci in confluent sheets when the surrounding cells are inhibited. These foci occur spontaneously in early-passage NIH 3T3 cells grown to confluency in 10% calf serum (CS) but are not seen in cultures grown to confluency in 2% CS. However, repeated passage of the cells at low density in 2% CS gives rise to an adapted population that grows to increasingly higher saturation densities and produces large numbers of foci in 2% CS. The increased saturation density of the adapted population in 2% CS is retained upon repeated passage in 10% CS, but the number and size of the foci produced in 2% CS gradually decrease under this regime. Clonal analysis confirms that the focus-forming potential of most if not all of the cells in a population increases in response to a continuously applied growth constraint, although only a small fraction of the population may actually form foci in a given assay. The acquired capacity for focus formation varies widely in clones derived from the adapted population and changes in diverse ways upon further passage of the clones. We propose that the adaptive changes result from progressive selection of successive phenotypic variations in growth capacity that occur spontaneously. The process designated progressive state selection resolves the apparent dichotomy between spontaneous mutation with selection on the one hand and induction on the other, by introducing selection among fluctuating states or metabolic patterns rather than among genetically altered cells.
Factors governing helical preference of peptides containing multiple alpha,alpha-dialkyl amino acids.
Authors: Marshall G R , Hodgkin E E , Langs D A , Smith G D , Zabrocki J , Leplawy M T
Published: May 2006

The presence of multiple alpha,alpha-dialkyl amino acids such as alpha-methylalanine (alpha-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with alpha-helical or 3(10)-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure alpha-helical character, whereas other similar compounds show predominantly 3(10)-helical structures. The factors that govern helical preference include the inherent relative stability of the alpha-helix compared with the 3(10)-helix, the extra hydrogen bond seen with 3(10)-helices, and the enhanced electrostatic dipolar interaction of the 3(10)-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions.
C-terminal truncation of the retinoblastoma gene product leads to functional inactivation.
Authors: Shew J Y , Lin B T , Chen P L , Tseng B Y , Yang-Feng T L , Lee W H
Published: May 2006

Mutational inactivation of the retinoblastoma (RB) gene has been implicated in the genesis of retinoblastoma, osteosarcoma, and other human tumors. Our strategy has been to characterize naturally occurring mutants from tumor cells to pinpoint potential domains of RB protein crucial for tumor suppression. We show here that osteosarcoma cell line Saos-2 contains an abnormal endogenous RB protein of 95 kDa (p95) that is located mainly in the cytoplasm. This protein was identified by antibodies recognizing several different RB epitopes, but not by one directed solely against the C terminus, suggesting C-terminal truncation. This conclusion was supported by analysis of mRNA and genomic DNA, which revealed that a transcriptionally active RB allele had a deletion of exons 21-27. In contrast to normal RB protein, this truncated protein was not phosphorylated and did not bind to the large tumor (T) antigen encoded by simian virus 40. We previously reported that introduction of normal RB protein into Saos-2 cells suppressed their neoplastic phenotype, indicating functional inactivation of their endogenous RB genes. These results provide an initial step to elucidate domains crucial to the cancer-suppression function of RB protein; its C-terminal portion is evidently important for this activity.
Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.
Authors: Warren S T , Knight S J , Peters J F , Stayton C L , Consalez G G , Zhang F P
Published: May 2006

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.
Human immunodeficiency virus type 1 expression in the central nervous system correlates directly with extent of disease.
Authors: Weiser B , Peress N , La Neve D , Eilbott D J , Seidman R , Burger H
Published: May 2006

To investigate human immunodeficiency virus type 1 (HIV-1) pathogenesis in infected individuals and examine the correlation of HIV-1 expression with extent of clinical and pathologic disease, we studied spinal cords from acquired immunodeficiency syndrome patients with a wide range of spinal cord pathology. By performing in situ hybridization with HIV-1-specific riboprobes, we detected HIV-1 RNA in all 10 cords from acquired immunodeficiency syndrome patients with a common, characteristic pathologic entity called vacuolar myelopathy but not in 10 control cords from HIV-1-infected and uninfected patients. In the cords from individuals with vacuolar myelopathy, the level of HIV-1 RNA expression correlated directly with extent of spinal cord pathology and clinical findings. These data support a role for HIV-1 in the pathogenesis of tissue damage and related clinical disease in infected individuals.
Sequence of an influenza virus hemagglutinin determined directly from a clinical sample.
Authors: Rajakumar A , Swierkosz E M , Schulze I T
Published: May 2006

The sequence of the HA1 region of the hemagglutinin gene of an influenza virus has been determined without growing the virus in eggs or in cultured cells. The virus used was an H1 strain of influenza A from a clinical specimen taken from a patient in 1987. RNA was extracted directly from virus that had been sedimented out of the transport medium in which the sample had been stored. DNA copies of the hemagglutinin gene, obtained by reverse transcription, were then amplified by the polymerase chain reaction and were sequenced by the dideoxy termination method. The deduced amino acid sequence is highly similar to that of other H1 viruses that had been isolated at about the same time and cultured for a limited number of passages in eggs. Furthermore, the HA1 sequence of progeny virus from this isolate obtained after one passage in chicken embryos is identical to that of the virus obtained directly from the nasopharynx. The results suggest that H1 isolates that have been grown for a limited number of passages in embryonated eggs have HA1 subunits that faithfully represent the virus population in the clinical samples from which they were derived.
Regularities in the sequences of the number of nucleons in the revolving clusters for the ground-state energy bands of the even-even nuclei with neutron number equal to or greater than 126.
Authors: Pauling L
Published: May 2006

Values of m, the number of nucleons in the revolving cluster, and of R, the radius of revolution of the cluster about the center of mass of the spherical part of the nucleus, are calculated from the observed values of the energy for the ground-state bands of all nuclei with neutron number N >/= 126 on the basis of the assumptions (i) that both m and R change in a reasonable way with increase in the angular momentum quantum number J and with change in the proton number Z and the neutron number N, (ii) that m is usually an even integer, (iii) that certain clusters are especially stable, and (iv) that there is a special stability of the doubly magic sphere p82n126.
Epithelial K channel expressed in Xenopus oocytes is inactivated by protein kinase C.
Authors: Sullivan S K , Swamy K , Greenspan N R , Field M
Published: May 2006

K homeostasis is maintained in higher animals by epithelia of the kidney and intestine. Little is known regarding the molecular regulation of K secretion. We injected Xenopus oocytes with mRNA from teleost intestine, a K-secreting epithelium with apical membrane K channels. Oocytes expressed a conductance that displayed whole-cell current properties with the following characteristics: marked selectivity for K over Na and Cl, voltage-independent kinetics, Ca insensitivity, tonic activation, and inward rectification in symmetrical K. Barium, quinine, and tetraethylammonium blocked the conductance, whereas apamin, charybdotoxin, and 4-aminopyridine did not. The K conductance was rapidly (t1/2 = 10 min) and completely inactivated by 4 beta-phorbol 12-myristate 13-acetate but not by 4 alpha-phorbol 12,13-didecanoate. Sucrose density gradient fractionation revealed that mRNA required for expression is in the 1- to 2-kilobase size range, suggesting the possibility that a single subunit encodes the channel. The K conductance expressed from injection of size-fractionated mRNA was identical in all respects to that seen using unfractionated mRNA, including response to 4 beta-phorbol 12-myristate 13-acetate. The results suggest that protein kinase C regulates K secretion in epithelia by modulation of apical K channels.