bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial separation of parental genomes in hybrids of somatic plant cells
Authors: Gleba Yuri Yu. , Parokonny A. , Kotov V. , Negrutiu I. , Momot V.
Published: May 2006

Chromosome spatial arrangements on metaphase plates of intergeneric intertribal cell hybrids of
Nicotiana chinensis
and
Atropa belladonna
as well as interspecific somatic hybrid plants of
Nicotiana plumbaginifolia
and
Nicotiana sylvestris
were analyzed. In the metaphases of the first divisions of protoplast fusion products, chromosomes of the two parents were spatially separated (segmented metaphase). In long-term cultured somatic hybrids, the topology of genome separation pattern in both callus cells and plants showed changes in form from “segmental” to “radial.” Growing the hybrid cells in the presence of colchicine resulted in random chromosome arrangement both in cells directly exposed to different colchicine concentrations and in colchicine-treated cells grown in colchicine-free media. The degree of genome separation calculated for different cell clones remained constant during
in vitro
propagation of cells but was significantly lower for subclones derived from colchicine-treated cells. Therefore, it is concluded that spatial chromosome arrangement in metaphase is epigenetically controlled.
Gap junction assembly in the preimplantation mouse conceptus is independent of microtubules, microfilaments, cell flattening, and cytokinesis.
Authors: Kidder G M , Rains J , McKeon J
Published: May 2006

Gap junctions first appear during compaction in the eight-cell stage of mouse development. Their assembly can be initiated in the near absence of transcription and protein synthesis from the four-cell stage, indicating the existence of preformed precursors. We have investigated the temporal control of this event, focusing on the possible involvement of the cytoskeleton, cell flattening, and cytokinesis. Embryos in various cleavage stages were treated with cytochalasins, to disrupt microfilaments and block cell flattening, cytokinesis, or both, or nocodazole, to promote microtubule depolymerization. To assess their capacity to initiate gap junction assembly after such treatments, the embryos were then aggregated with communication-competent, compacted embryos that had been labeled with carboxyfluorescein diacetate. Passage of the fluorescent dye, carboxyfluorescein, from labeled to unlabeled embryo was taken as evidence that interembryonic junction formation had occurred. The capacity to assemble gap junctions was acquired at the normal time by embryos prevented by cytochalasin treatment from undergoing cell flattening or any cytokinesis from fertilization onward. Likewise, treatment with nocodazole beginning in the four-cell or early eight-cell stage did not interfere with gap junction assembly. Neither drug affected the inability of four-cell embryos to assemble gap junctions prematurely. We conclude that intact microfilament or microtubule networks are not required for gap junction assembly in this system, nor do they restrain junctional precursors from assembling prematurely. Furthermore, the timing of gap junction assembly is not linked to cell flattening, cytokinesis, or cell number.
Prostaglandin F2 alpha stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca2+ in bovine luteal cells.
Authors: Davis J S , Weakland L L , Weiland D A , Farese R V , West L A
Published: May 2006

The present studies were conducted to determine whether prostaglandin F2 alpha (PGF2 alpha) stimulates the production of "second messengers" derived from inositol phospholipid hydrolysis and increases intracellular free Ca2+ ([Ca2+]i) in isolated bovine luteal cells. PGF2 alpha provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (InsP, InsP2, and InsP3, respectively). InsP3 was formed more rapidly than InsP2 or InsP after PGF2 alpha treatment. In addition, PGF2 alpha increased inositol phospholipid turnover, as evidenced by increased 32PO4 incorporation into phosphatidic acid and phosphatidylinositol. LiCl (1-20 mM) enhanced inositol phosphate accumulation in response to PGF2 alpha. Maximal increases in InsP3 occurred at 1 microM PGF2 alpha, with half-maximal stimulation occurring at 36 nM. The acute effects of PGF2 alpha on InsP3 levels were independent of reductions in extracellular calcium. Prostaglandins E1 and E2 also stimulated increases in inositol phosphate levels, albeit to a lesser extent. PGF2 alpha also induced rapid and concentration-dependent increases in [Ca2+]i as measured by quin-2 fluorescence. The PGF2 alpha-induced increases in [Ca2+]i were maximal within 30 sec (approximately 2- to 3-fold), and [Ca2+]i remained elevated for 8-10 min. The PGF2 alpha-induced increases in [Ca2+]i were also independent of extracellular calcium. These findings demonstrate that the action of PGF2 alpha is coupled to the phospholipase C-InsP3 and diacylglycerol second messenger system in the corpus luteum.
Isolation of a T-cell clone that reacts with both antigen and anti-idiotype: evidence for anti-idiotype as internal image for antigen at the T-cell level.
Authors: Singhai R , Levy J G
Published: May 2006

T-cell lines were derived from ferredoxin nonresponder B10.D2 mice that share an idiotype expressed by a monoclonal antibody (Fd-B2) with specificity for one of the two major antigenic determinants (the C determinant) of the antigen. The T-cell line and T-cell clones derived from it release interleukin 2 not only in the presence of anti-Fd-B2 idiotype antibody but in the presence of ferredoxin. The line was shown to be major histocompatibility complex-restricted in that it would respond to the anti-idiotype and antigen only in the context of presentation by cells of the H-2d haplotype. This observation also establishes that the nonresponder status of H-2d animals cannot be attributed to a lesion at the level of antigen presentation. Analysis of the fine specificity of one idiotypic clone showed that it responded only to the anti-idiotype or products of the antigen containing the C determinant, since enzymatically degraded peptides devoid of this determinant did not stimulate these cells. Furthermore, it was found that presentation of both the antigen and the anti-idiotype to the specific clone could be blocked by the Fd-B2 monoclonal antibody.
Stable transformation of soybean by electroporation and root formation from transformed callus
Authors: Christou Paul , Murphy Jean E. , Swain William F.
Published: May 2006

Soybean protoplasts from a number of commercially important cultivars have been genetically engineered by way of electroporation using chimeric genes coding for resistance to the aminoglycoside antibiotics kanamycin and G418. Effective electroporation conditions were determined by monitoring transient expression from aminoglycoside 3′-phosphotransferase II (APHII) expression plasmids. Electroporation of protoplasts with a chimeric APHII gene and subsequent selection on media supplemented with kanamycin resulted in the recovery of calli resistant to the antibiotic. Enzyme assays for APHII activity and Southern blot hybridization confirmed the expression of the foreign DNA and its stable integration into the soybean genome. Root formation was induced from transformed calli, and these roots maintained expression of the APHII gene.
cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe).
Authors: Glasser S W , Korfhagen T R , Weaver T , Pilot-Matias T , Fox J L , Whitsett J A
Published: May 2006

Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of Mr 6000-14,000 in immunoblot analysis and was used to screen a lambda gt11 expression library constructed from adult human lung poly(A)+ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused beta-galactosidase-SPL(Phe) gene in Escherichia coli yielded an immunoreactive Mr 34,000 fusion peptide. Hybrid-arrested translation with this cDNA and immunoprecipitation of [35S]methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a Mr 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. The larger RNA and translation product indicates that SPL(Phe) is derived by proteolysis of a large polypeptide precursor. The amino acid sequence of the predicted protein, beginning Phe-Pro-Ile-Pro-Leu-Pro-Try-, comprises a hydrophobic peptide that is a major protein component of surfactant lipid extracts used successfully to treat hyaline membrane disease in newborn infants. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.
Lipid monolayer states and their relationships to bilayers.
Authors: MacDonald R C , Simon S A
Published: May 2006

Uncommon methods of formation and analysis of lipid monolayers have enabled the recognition of several monolayer states and the identification of that in which molecular organization corresponds closely to that of the bilayer. Monolayers were formed by continuously adding a solution of phospholipid [dimyristoyl phosphatidylcholine in hexane/ethanol, 9:1 (vol/vol)] to the air/water interface of a constant-area trough. This procedure generates unconventional surface pressure (pi)-surface concentration (gamma) isotherms, which for liquid-crystalline monolayers consist of straight lines with three prominent intersections, two of which are not apparent in conventional pi-A isotherms. The regions of linear change of pi are explicable in terms of the area dependence of alkyl chain entropy. The two breaks at lower pi delimit states in which both chains lie parallel to the surface. The third occurs at collapse, which corresponds to a true equilibrium for unstressed liposomes. Mechanical and thermodynamic properties of bilayers, particularly phase-transition parameters, correspond closely to those of monolayers with which they are in equilibrium.
Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin
Authors: Corcoran Timothy C. , Ismail Kamal Z. , El-Sayed Mostafa A.
Published: May 2006

The removal of metal cations inhibits the deprotonation process of the protonated Schiff base during the photocycle of bacteriorhodopsin. To understand the nature of the involvement of these cations, a spectroscopic and kinetic study was carried out on bacteriorhodopsin samples in which the native Ca
2+
and Mg
2+
were replaced by Eu
3+
, a luminescent cation. The decay of Eu
3+
emission in bacteriorhodopsin can be fitted to a minimum of three decay components, which are assigned to Eu
3+
emission from three different sites. This is supported by the response of the decay components to the presence of
2
H
2
O and to the changes in the Eu
3+
/bR molar ratio. The number of water molecules coordinated to Eu
3+
in each site is determined from the change in its emission lifetime when
2
H
2
O replaces H
2
O. Most of the emission originates from two “wet” sites of low crystal-field symmetry—e.g., surface sites. Protonated Schiff base deprotonation has no discernable effect on the emission decay of protein-bound Eu
3+
, suggesting an indirect involvement of metal cations in the deprotonation process. Adding Eu
3+
to deionized bacteriorhodopsin increases the emission intensity of each Eu
3+
site linearly, but the extent of the deprotonation (and color) changes sigmoidally. This suggests that if only the emitting Eu
3+
ions cause the deprotonation and bacteriorhodopsin color change, ions in more than one site must be involved—e.g., by inducing protein conformation changes. The latter could allow deprotonation by the interaction between the protonated Schiff base and a positive field of cations either on the surface or within the protein.
SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth.
Authors: Craig E A , Kramer J , Kosic-Smithers J
Published: May 2006

The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes. Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effects of the mutation in SSD1 were detected. However, a functional SSC1 gene is essential for vegetative growth. This result, in conjunction with experiments involving mutations in other members of this multigene family, indicates that at least three distinct functions are carried out by genes of the HSP70 family.
Rearrangement and expression of rabbit immunoglobulin kappa light chain gene in transgenic mice.
Authors: Goodhardt M , Cavelier P , Akimenko M A , Lutfalla G , Babinet C , Rougeon F
Published: May 2006

To determine whether a foreign unrearranged immunoglobulin gene can be functionally rearranged and expressed in vivo, a rabbit b9 kappa light chain gene construct containing a single germ-line kappa chain variable (V) region gene (V kappa), the five kappa chain joining (J) segments (J kappa), and the kappa chain constant (C) region germ-line gene (C kappa) was introduced into fertilized mouse eggs. Eleven transgenic mice carrying 1-30 copies of the rabbit kappa gene construct were obtained. Rearrangement of the transgene due to V kappa-J kappa recombination was observed in the spleen of all the mice lines analyzed. Only the J kappa 1 and J kappa 2 segments, which have canonical sequences for rearrangement and high-level expression, are utilized in assembly of the rabbit gene. V kappa-J kappa 1 and V kappa-J kappa 2 joining was also observed in the thymus but not in nonlymphoid tissue. Polyadenylylated rabbit kappa chain transcripts of 1.2 kilobases were found in the spleen of the transgenic mice. The level of transcription was low despite a high level of rearrangement. Three transgenic mice lines secreted kappa light chains encoded by the foreign rabbit gene. Serum rabbit kappa chains were associated with mouse mu and gamma 1 heavy chains. However, hybrid antibody molecules containing both rabbit and mouse kappa light chains were also found in the serum of these animals. These results suggest that, although the rabbit kappa chain gene construct contains the necessary sequences for gene assembly, sequences implicated in stage- and tissue-specific regulation of kappa chain gene rearrangement are either missing or not recognized by mouse lymphoid cells.
Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vaccinated mice.
Authors: Dalton J P , Tom T D , Strand M
Published: May 2006

Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambda gt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.
The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors.
Authors: Kettelhut I C , Fiers W , Goldberg A L
Published: May 2006

Tumor necrosis factor (TNF) is a macrophage product under active study as an anticancer drug. However, this agent can be very toxic and has been implicated in the pathogenesis of endotoxic shock. After intravenous injection of human recombinant TNF (4 micrograms/g), growing rats showed an unusual constellation of physiological responses, and all died within 2-4 hr. In 1 hr, TNF caused a sharp fall (2.5 degrees C) in body temperature and a large increase in plasma prostaglandin E2 levels. Blood glucose initially increased, but then a profound hypoglycemia developed by 2 hr. The TNF-treated animals also showed diarrhea, cyanosis, and a severe metabolic acidosis. A single injection of the cyclooxygenase inhibitors indomethacin or ibuprofen before the TNF treatment completely prevented the rapid killing and reduced eventual lethality by 70%. These agents blocked prostaglandin E2 production and prevented the hypothermia, changes in blood glucose, acidosis, and other symptoms. Since similar physiological changes have been reported after endotoxin injection, our data support the suggestion that TNF production is a critical factor in the development of septic shock. These findings also indicate that increased production of prostaglandins or thromboxanes is important in endotoxic shock and argue that cyclooxygenase inhibitors should be useful in its therapy. Indomethacin did not block the cytotoxic effects of TNF in vitro on several transformed cell lines (HeLa, Me 180, or L929). Therefore, combined use of TNF with a cyclooxygenase inhibitor may allow safer administration of high doses of this polypeptide to cancer patients.
Identification of residues required for ligand binding to the beta-adrenergic receptor.
Authors: Strader C D , Sigal I S , Register R B , Candelore M R , Rands E , Dixon R A
Published: May 2006

The functional significance of conserved polar amino acids within the putative transmembrane region of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding beta AR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant beta AR did not show detectable binding toward the antagonist iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the beta AR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the beta AR.
Theoretically determined three-dimensional structure for the repeating tetrapeptide unit of the circumsporozoite coat protein of the malaria parasite Plasmodium falciparum.
Authors: Brooks B R , Pastor R W , Carson F W
Published: May 2006

A model for the three-dimensional structure of the repeating Asn-Pro-Asn-Ala tetrapeptide of the immunodominant circumsporozoite protein of Plasmodium falciparum has been developed. A trial structure in the form of a type I beta turn with asparagine side chains hydrogen-bonded to the backbone peptide linkages was used as a starting point. A repeating oligomer of this trial structure was modeled using energy minimization and molecular dynamics computer simulations in conjunction with image boundary conditions. The most stable structure generated is a right-handed 12(38) helix, which is unlike any previously identified protein secondary structure. The helix has 12 residues per turn, corresponding to an angle of twist of 120 degrees per tetrapeptide unit, and a pitch of 4.95 A, corresponding to a rise of 1.65 A per tetrapeptide unit. It is highly stabilized by extensive hydrogen bonding, with each tetrapeptide unit acting as an acceptor for five hydrogen bonds and as a donor for five hydrogen bonds to residues in adjacent turns as well as having four weak internal hydrogen bonds. A number of nearly isoenergetic variations on the most stable structure that still retained the basic 12(38) helical motif were also discovered. The implications of these structures for vaccine development are discussed.
Genetic instability in Drosophila melanogaster: cytogenetic analysis of MR-induced X-chromosome deficiencies.
Authors: Green M M , Yamamoto M T , Miklos G L
Published: May 2006

We present data that demonstrate that three MR elements isolated from wild populations of Drosophila melanogaster on two continents can cause large deletions of the X chromosome in males. The deleted chromosomes, termed mini-X chromosomes, are induced at a frequency of approximately 1:4000 in chromosomes that are initially free of P elements. In situ hybridizations using a cloned P sequence as a probe fail to reveal any sequences homologous to the nomadic P family at the deletion breakpoints. Genetic analysis of 12 such mini-X chromosomes also reveals that there are no "hotspots" of chromosome breakage and that there must have been a minimum of three distinct distal breakpoints and five different proximal breakpoints in the formation of these deleted chromosomes. In fact all 12 proximal and 12 distal breakpoints may well be unique. Our data show that MR elements generate essentially random breaks along the X chromosome. We emphasize that we find no involvement of P sequences in the chromosome breakage process, consonant with the notion that MR elements exert their influence on processes involved in mitotic crossing-over.
HLA-DR2 subtypes form an additional supertypic family of DR beta alleles.
Authors: Lee B S , Rust N A , McMichael A J , McDevitt H O
Published: May 2006

Homozygous lymphoblastoid cell lines representing Dw subtypes of the DR2 serotype were studied for structural polymorphism at DR beta. These subtypes included Dw2, Dw12, and non-Dw2/non-Dw12. Analysis by two-dimensional gel electrophoresis showed that two DR beta genes were expressed in each cell line studied. One of these expressed genes encoded a protein that was nonpolymorphic on two-dimensional gel electrophoresis among all subtypes. The second expressed DR beta gene was polymorphic and migrated to a position on two-dimensional gels dependent on the subtype of the cell line. cDNA sequence analysis of the DR beta genes revealed a DR beta gene identical between the Dw2 and Dw12 subtypes, which correlates with the nonpolymorphic spot on two-dimensional gels. A second gene was sequenced that exhibited variability between the Dw2 and Dw12 subtypes. This variability takes the form of clustered point mutations in the first domain of the molecules. The alleles from the DR beta genes of a non-Dw2/non-Dw12 cell line, AZH, were unusual in sequence. In contrast to other DR beta alleles, the AZH genes may have been generated by a double recombinational event between two DR beta loci from a DR2 parent. The DR2 serotype may also constitute a supertypic "family," with one DR beta gene relatively nonpolymorphic, and one that varies with Dw subtype.
Murine B-cell stimulatory factor 1 (interleukin 4) increases expression of the Fc receptor for IgE on mouse B cells.
Authors: Hudak S A , Gollnick S O , Conrad D H , Kehry M R
Published: May 2006

We have studied the activity of mouse B-cell stimulatory factor 1 (interleukin 4, IL-4) on resting splenic B cells and on a B-cell hybridoma. Purified T-cell-derived as well as recombinant IL-4 was shown to increase the expression of the low-affinity Fc receptor for IgE (Fc epsilon R) on a majority of B lymphocytes in a 24-hr culture period. Levels of Fc epsilon R expression increased 2- to 3-fold on splenic B cells and up to 6-fold on a B-cell hybridoma. The effect was inhibited by an anti-IL-4 monoclonal antibody and by mouse gamma-interferon. Other recombinant lymphokines exhibited no effect on either Fc epsilon R expression or the induction by IL-4. The presence of IgE during the stimulation with IL-4 resulted in an additional increase in Fc epsilon R expression. These data and results showing that IgE prevents Fc epsilon R turnover while IL-4 increases the rate of Fc epsilon R synthesis suggest that the mechanisms by which IgE and IL-4 increase Fc epsilon R expression are likely to be different. The starting population of splenic B cells expressed low levels of Fc epsilon R and was relatively uniform in size (small). After greater than 48 hr of culture with IL-4, viable B cells had not undergone DNA synthesis and consisted mainly of larger highly Fc epsilon R-positive cells (23%) and medium-sized Fc epsilon R-positive cells (60%). A possible role for Fc epsilon R in certain B-cell maturation pathways is discussed.
Structural effects on surfaces within layered crystals
Authors: McBride J. Michael , Bertman Steven B. , Semple Thomas C.
Published: May 2006

Within a series of derivatives of diundecanoyl peroxide it has been possible to compare a variety of bulk and molecular-level properties among structures that differ only in a region at the interface between crystal lamellae. This region is small enough to allow reliable structure/property correlation. The derivatives involve halogenation and variation of chain length. The properties include crystal structure, melting point (odd-even alternation), thermal expansion, compressibility, and accommodation of interstitial molecules (as measured by the vibrational frequency of CO
2
impurity). In general the properties reflect the efficiency of geometric packing at the interface, with halogen substituents exerting additional nongeometric influence.
Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells.
Authors: Levis R , Huang H , Schlesinger S
Published: May 2006

We are investigating the feasibility of using the positive-strand RNA virus Sindbis virus and its defective interfering (DI) particles as vectors for introducing foreign genes into cells. In previous work we showed by deletion mapping of a cloned cDNA derived from one of the DI RNAs that only nucleotides at the 3' and 5' termini of the RNA are essential for the DI RNA to be amplified after it is transfected into cells in the presence of helper virus. As a first step in developing a vector we replaced 75% of the internal nucleotides of this DI cDNA with foreign sequences including the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. DI RNAs transcribed from this cDNA were replicated and packaged by helper Sindbis virus and became a major viral RNA species in infected cells by the third passage after transfection. They were also translated to produce enzymatically active CAT. CAT activity was measured at passage 3 but could also be detected in transfected cells. DI RNAs containing the CAT gene were translated in vivo and in vitro to produce two polypeptides immunoprecipitable by anti-CAT antibodies. One polypeptide was identical in size to the authentic CAT polypeptide; the other was the size expected for a protein initiated at an upstream, viral-specific AUG in frame with the CAT AUG. These studies establish that DI genomes of Sindbis virus can tolerate the insertion and direct the expression of at least one foreign gene.
Metal-dependent folding of a single zinc finger from transcription factor IIIA.
Authors: Frankel A D , Berg J M , Pabo C O
Published: May 2006

A 30-amino acid peptide, which corresponds to the second "zinc finger" domain of transcription factor IIIA, has been synthesized and purified. This peptide folds in the presence of zinc: adding Zn2+ significantly changes the circular dichroism spectrum, and Zn2+ protects the peptide from tryptic digestion. The peptide also binds Co2+, and the absorption spectrum of the Co2+ complex suggests that a tetrahedral binding site is formed by two cysteines and two histidines. Experiments at higher temperatures (60-75 degrees C) suggest that these folded metal-peptide complexes are quite thermostable. The peptide shows some sequence-specific effects in DNase and methylation protection experiments. However, it does not give a clear "footprint," and some effects are observed in the absence of added zinc.
Monoclonal antibody specific for the transverse tubular membrane of skeletal muscle activates the dihydropyridine-sensitive Ca2+ channel.
Authors: Malouf N N , Coronado R , McMahon D , Meissner G , Gillespie G Y
Published: May 2006

In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive Ca2+ channels are preferentially localized in the transverse tubular membranes. Starting with an antigenic membrane fraction enriched in rabbit muscle transverse tubules (T-tubules), several monoclonal antibodies were produced by a fusion of spleen cells from an immunized BALB/c mouse with P3 X 63Ag.8.6.5.3 mouse myeloma cells. Antibodies were screened according to a scheme designed to select IgG immunoglobins that recognized a determinant specifically associated with the T-tubule membrane. Antibodies that fulfilled the screening criteria were used in in vitro planar bilayer recording of the activity of the dihydropyridine-sensitive Ca2+ channel present in T-tubules. Cells producing one antibody (Ab 21) survived cloning dilution and stably produced a monoclonal antibody (mAb21-4) that increased the rate of single channel opening when interacting with the internal side of the channel protein. mAb21-4 immobilized by covalent crosslinking on beads (Affi-Gel 10) consistently immunoprecipitated polypeptide bands with the following electrophoretic mobility: Mr values of greater than or equal to 175,000; 90,000; 55,000; and 34,000.
4-Spiro[2.n]alkyl cations and their rearrangements.
Authors: Prakash G K , Fung A P , Olah G A , Rawdah T N
Published: May 2006

A series of 4-spiro[2.n]alkanols, where n = 3-7, were ionized in either FSO3H x SbF5/SO2ClF or SbF5/SO2ClF at -78 degrees C and -130 degrees C. The resulting solutions were studied by 13C NMR spectroscopy over the temperature range of -130 degrees C to -10 degrees C. The 4-spiro[2.5]octanol gave the expected static 4-spiro[2.5]octyl cation, which can be considered as a long-lived secondary cyclohexyl cation stabilized by an adjacent spirocyclopropane ring. The same spiro[2.5]octyl cation was also obtained by ionization of isomeric bicyclo[4.2.0]octan-1-ol and bicyclo[4.1.0]heptyl-1-methanol. The static spiro[2.5]-octyl cation was found to rearrange to the equilibrating 1-bicyclo[3.3.0]octyl cation above -10 degrees C. On the other hand, the 4-spiro[2.4]heptanol gave the rearranged 1-methylcyclohexenyl cation. The spiro[2.6]nonanol gave directly the equilibrating 1-bicyclo[4.3.0]nonyl cation. The other 4-spiro[2.n]-alkanols (n = 3 and 7) gave unidentifiable polymeric products. The ionization of 3-spirocyclopropyl-2-norbornanol yields only the rearranged 2-methylbicyclo[3.2.1]oct-3-ene-2-yl cation.
The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain.
Authors: Scott J D , Glaccum M B , Zoller M J , Uhler M D , Helfman D M , McKnight G S , Krebs E G
Published: May 2006

A cDNA clone for a type II regulatory (R) subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from a rat skeletal muscle library using a specific 47-base oligonucleotide probe. The rat cDNA was 1.2 kilobases (kb) in length and contained an open reading frame of 1.113 kb representing 92% of the coding region of the molecule. Nick-translated rat cDNA was then used to isolate a mouse RII cDNA clone from a brain library that contained an open reading frame of 1.143 kb. Because both cDNAs lacked complete coding sequences, the remainder of the RII coding region was obtained from a 15-kb mouse genomic clone. The mouse RII coding region contains 1.2 kb corresponding to a 400-amino acid protein of 51.141 kDa. The mouse cDNA hybridizes to two mRNA species, a 2.4-kb form that was only observed in testis and a 6.0-kb form found in a wide range of tissues, including testis.
A common origin for enzymes involved in the terminal step of the threonine and tryptophan biosynthetic pathways.
Authors: Parsot C
Published: May 2006

Comparison of the amino acid sequence of Bacillus subtilis threonine synthase with the National Biomedical Research Foundation protein sequence library revealed a statistically significant extent of similarity between the sequence of the tryptophan synthase beta chain from various organisms and that of threonine synthase. This homology in the primary structure of threonine synthase and tryptophan synthase beta chain, which catalyze the last step in the threonine and the tryptophan biosynthetic pathways, respectively, correlates well with some of their catalytic properties and indicates that they have evolved from a common ancestor. The evolutionary relationship between these enzymes supports the hypothesis that primitive enzymes possessed a broad substrate specificity and were active in several metabolic pathways.
T-DNA organization in tumor cultures and transgenic plants of the monocotyledon
Asparagus officinalis
Authors: Bytebier Benny , Deboeck Francine , De Greve Henri , Montagu Marc Van , Hernalsteens Jean-Pierre
Published: May 2006

Asparagus officinalis
was the first monocotyledonous plant from which hormone-independent and opine-producing crown gall tissue could be isolated. We confirm by DNA hybridization that tumor lines obtained after infection of this plant by
Agrobacterium
strains harboring wild-type nopaline and octopine tumor-inducing (Ti) plasmids are stably transformed and contain transferred DNA (T-DNA) segments identical to the T-DNA found in dicotyledonous plants. We have also infected
Asparagus
with a nononcogenic T-DNA vector that carries a chimeric aminoglycoside phosphotransferase [NOS-APH(3′)II] gene and selected transformed tissues on kanamycin-containing medium. The transformed status of these tissues was then confirmed by DNA hybridization. From these calli we regenerated kanamycin-resistant shoots that were subsequently rooted. Thus we report the isolation of transgenic monocotyledonous plants engineered via the
Agrobacterium
vector system.
A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize
Authors: Dewey R. E. , Timothy D. H. , Levings C. S.
Published: May 2006

Mitochondrial DNA in the T male-sterile cytoplasm (cms-T) of maize contains an open reading frame (ORF 13) associated with the T type of sterility. Antibodies raised to a chemically synthesized oligopeptide corresponding to ORF 13 were used to establish the expression of a 13-kDa protein from this reading frame. The 13-kDa polypeptide is synthesized uniquely in cms-T maize and purifies with the membrane fraction of T mitochondria. We assign the symbol
urf13-T
to designate this mitochondrial gene. Presence of the nuclear restorer gene
Rf1
in cms-T plants results in a decrease in abundance of 13-kDa protein and alteration in the transcripts of
urf13-T
.
Nonrandom development of immunologic abnormalities after infection with human immunodeficiency virus: implications for immunologic classification of the disease.
Authors: Zolla-Pazner S , Des Jarlais D C , Friedman S R , Spira T J , Marmor M , Holzman R , Mildvan D , Yancovitz S , Mathur-Wagh U , Garber J
Published: May 2006

Blood specimens from 165 intravenous drug users who were seropositive for the human immunodeficiency virus (HIV), from 158 seropositive homosexual men with lymphadenopathy, and from 77 patients with acquired immunodeficiency syndrome (AIDS) were assessed immunologically. Immunologic parameters were analyzed by the Guttman scalogram technique to determine if immunologic abnormalities occurred in a nonrandom pattern. The following four patterns emerged: (i) seropositivity for HIV with no immunologic abnormalities; (ii) seropositivity for HIV with a depressed T4/T8 cell ratio; (iii) seropositivity with a depressed T4/T8 cell ratio and T4-cell depletion; and (iv) seropositivity with a depressed T4/T8 cell ratio, T4-cell depletion, and lymphopenia. Ninety-two to 100% of subjects in each of the three groups of patients were found "to scale" because the abnormalities occurred in the cumulative, ordered fashion described. This nonrandom occurrence of abnormalities indicates an ordered progression of immunologic abnormalities in individuals infected with HIV, a finding useful in the staging of both symptomatic and asymptomatic HIV-seropositive subjects.
Cannabinoids induce incomplete maturation of cultured human leukemia cells.
Authors: Murison G , Chubb C B , Maeda S , Gemmell M A , Huberman E
Published: May 2006

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 microM delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody or the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 microM THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. Pronounced among these changes was an increase in the synthesis of at least 10 proteins that are found abundantly in monocytes. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype; the THC-treated cells failed to exhibit other monocyte markers such as attachment to the surface of tissue culture dishes or morphological maturation beyond the promonocyte stage. However, treatment of these "incompletely" matured cells with either phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. Two other cannabinoids, cannabidiol and cannabinol, which were more cytotoxic than THC at comparable doses, also caused an increase in the expression of maturation markers, but at doses higher than those required for THC. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced "incomplete" cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.
Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor.
Authors: Pohlmann R , Nagel G , Schmidt B , Stein M , Lorkowski G , Krentler C , Cully J , Meyer H E , Grzeschik K H , Mersmann G
Published: May 2006

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in lambda gt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids (Mr = 30,993), and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (Mr = 18,299) and C-terminal (Mr less than or equal to 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12.
Molecular cloning of a prolactin-related mRNA expressed in bovine placenta.
Authors: Schuler L A , Hurley W L
Published: May 2006

Bovine (Bos taurus) prolactin-related cDNA I (bPRC-I), distinct from the isolated bovine placental lactogen, was derived from bovine fetal placental mRNA by molecular cloning. The nucleotide sequence is 63% homologous to bovine prolactin cDNA and only 45% to bovine growth hormone. The region of bPRC-I corresponding to the 5' portion of the signal peptide and 5' untranslated region of bovine prolactin mRNA is markedly different from prolactin. The predicted protein is 39% homologous to bovine prolactin and about 30% to the related placental hormones in rodents. This identification of a prolactin-related gene in the cow in addition to those reported in rodents suggests that multiple prolactin-related genes expressed in the placenta may be a general phenomenon in nonprimates. The role of these related hormones during gestation remains to be investigated.
Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus.
Authors: Emerson S U , Schubert M
Published: May 2006

Recombinant DNA techniques were used to delete regions of a cDNA clone of the phosphoprotein NS gene of vesicular stomatitis virus. The complete NS gene and four mutant genes containing internal or terminal deletions were inserted into a modified pGem4 vector under the transcriptional control of the phage T7 promoter. Run-off transcripts were synthesized and translated in vitro to provide [35S]methionine-labeled complete NS or deletion mutant NS proteins. Immune coprecipitation assays involving these proteins were developed to map the regions of the NS protein responsible for binding to the structural viral nucleocapsid protein N and the catalytic RNA polymerase protein L. The data indicate the NS protein is a bivalent protein consisting of two discrete functional domains. Contrary to previous suggestions, the negatively charged amino-terminal half of NS protein binds to L protein, while the carboxyl-terminal half of NS protein binds to both soluble recombinant nucleocapsid protein N and viral ribonucleocapsid template.
Expression and nuclear envelope localization of biologically active fusion glycoprotein gB of herpes simplex virus in mammalian cells using cloned DNA.
Authors: Ali M A , Butcher M , Ghosh H P
Published: May 2006

Herpes simplex virus (HSV) is known to bud from the inner membrane of the nuclear envelope. The structural gene for the glycoprotein gB, which is essential for virus entry and cell fusion induced by HSV type 1, has been cloned in a transient expression vector containing the adenovirus major late promoter, tripartite leader sequence, and VARNA (a special RNA in adenovirus-infected cells) genes. Synthesis and glycosylation of glycoprotein gB was observed in COS-1 cells transfected with the vector containing the gB gene. Removal of a 3' fragment of the cloned gene resulted in the synthesis and secretion of a truncated gB glycoprotein. Immunofluorescence studies revealed that the expressed glycoprotein was localized in the nuclear envelope as well as in the cell surface. The expressed gB-1 glycoprotein was biologically active and induced fusion of cells to produce polykaryons. These data show that HSV glycoprotein gB expressed from cloned gene can be used as a model to study targeting of proteins into the nuclear envelope as well as cell fusion induced by the virus.
Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors.
Authors: Allen J P , Feher G , Yeates T O , Komiya H , Rees D C
Published: May 2006

The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 A with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure.
Construction of a Leydig cell line synthesizing testosterone under gonadotropin stimulation: a complex endocrine function immortalized by cell hybridization.
Authors: Finaz C , Lefèvre A , Dampfhoffer D
Published: May 2006

Hybridization between a mouse Leydig tumor cell line, MA-10, which produces cyclic AMP and progesterone under human chorionic gonadotropin (hCG) stimulation, and freshly isolated mouse Leydig cells gave rise to 54 hybrid clones, one of which, LK17, was capable of hCG-stimulated testosterone production. Subcloning of this hybrid resulted in the emergence of a subclone, K9, whose testosterone production is more than 10 times that of parent clone LK17, after hCG stimulation, with an ED50 of 37 pM. Testosterone synthesis by K9 cells was multiplied by 25 after gonadotropin stimulation, and binding of hCG declined after prolonged exposure to the hormone. These similarities with murine Leydig cells in primary culture make the K9 clone an attractive alternative for physiological studies.
Multiple topogenic sequences in bovine opsin.
Authors: Audigier Y , Friedlander M , Blobel G
Published: May 2006

Bovine opsin, a polytopic integral membrane protein, contains seven transmembrane segments connecting eight hydrophilic domains alternating on each side of the membrane. To localize topogenic sequences that might specify the distinct topology of opsin in the membrane, we constructed various opsin mutants, each containing only one transmembrane segment. Messenger RNAs transcribed from these mutants were translated in a cell-free system supplemented with microsomal membranes. Among six of the seven transmembrane segments of opsin that were analyzed, five were able to function as signal sequences and also expressed stop-transfer sequences of variable strength. By the criteria of extractability at pH 11 and protease sensitivity, the presence of a signal sequence in combination with a "strong" stop-transfer sequence yielded integration into the lipid bilayer of the majority of chains. However, in combination with a "weak" stop-transfer sequence, we observed integration into the lipid bilayer of only some chains, with the others either completely translocated across the membrane or retained in a water-accessible space in the membrane.