bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6.
Authors: Haruta Y , Seon B K
Published: May 2006

In this study, a monoclonal antibody (mAb) termed SN6 was generated by immunizing a mouse with a non-T-cell leukemia antigen preparation isolated from cell membranes of leukemia cells derived from a patient (FJ) with non-T/non-B-cell-type acute lymphoblastic leukemia (ALL). SN6 was tested against a variety of cultured and uncultured human cell specimens by using a sensitive cellular radioimmunoassay. Among the 26 cultured malignant and nonmalignant cell lines tested, SN6 reacted with all of the 6 leukemic non-T/non-B (including pre-B)-cell lines tested--i.e., KM-3, NALM-16, REH, NALL-1, NALM-1, and NALM-6. Of these cell lines, 5 were derived from individual patients with ALL; the remaining 1 was from a patient with chronic myelocytic leukemia in blast crisis. In addition, SN6 reacted with 3 of 3 leukemic myelo-monocytic cell lines tested--i.e., ML-2, HL-60, and U937. SN6 did not react with any other cell lines. A consistent result was obtained with 42 fresh (uncultured) cell specimens derived from individual patients with several different types of leukemias. SN6 reacted with 11 of 16 non-T/non-B (including pre-B)-cell ALL specimens. In addition, it reacted with various myelo-monocytic leukemia cell specimens to various degrees. SN6 did not show a significant reaction with normal peripheral blood cells tested, which included B cells, T cells, granulocytes, monocytes, and erythrocytes. However, it reacted with a small population (approximately 1% as determined by immunofluorescence staining) of normal bone marrow cells. The approximate molecular mass of the glycoprotein antigen defined by SN6 was determined to be 160,000 by radioimmunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Only one component of 80,000 daltons was formed upon reduction of the 160,000 molecular mass antigen. Therefore, this antigen is apparently a homodimer of a 80,000-dalton subunit. This conclusion was further corroborated by two-dimensional gel analysis, which showed a single well-defined spot for the reduced antigen. We designate this distinct human leukemia-associated cell surface antigen "GP160."
Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays.
Authors: Suresh M R , Cuello A C , Milstein C
Published: May 2006

A chemical selection procedure has been used to prepare a hybrid hybridoma cell line (P4C1) following fusion of two previously established hybridomas secreting antiperoxidase and antisubstance P, respectively. P4C1 secretes bispecific monoclonal antibody alongside the two parental antibodies, with no visible inactive heterologous heavy-light chain pairs. The bispecific monoclonal antibody is thus easy to purify in excellent yields. The advantage of its monovalency for one antigen and simultaneous binding of a marker enzyme has been explored for its potential use in competitive immunoassays. Its use in immunocytochemistry led to major improvements in sensitivity, signal-to-noise ratio, simplification of staining procedures, and ultrastructural preservation of subcellular elements. Particularly remarkable was that, unlike conventional procedures, the immunoreaction with the bispecific monoclonal antibody was homogeneously distributed across the entire thickness of a 50-micron section.
Human striatal dopamine receptors are organized in compartments.
Authors: Joyce J N , Sapp D W , Marshall J F
Published: May 2006

Dopamine (D2) receptors visualized in postmortem human striatum by quantitative autoradiography of [3H]spiroperidol binding are organized into circumscribed zones of low receptor density separated from other such zones by regions of higher D2 density. The D2-rich zones of the caudate nucleus and putamen contain twice the binding of D2-poor zones. The Hill coefficient, obtained from saturation analysis of [3H]spiroperidol binding to thin sections of human striatum, gave a value near unity, indicating the binding was occurring to a single type of site. The patchiness of [3H]spiroperidol binding was unaltered by postincubation removal of lipid from the tissue sections, indicating that a differential absorption of tritium in white and grey matter does not account for the heterogeneous distribution. The D2-rich and D2-poor regions appear to form labyrinths oriented in the anterior-posterior axis and are typically aligned with, respectively, acetylcholinesterase-rich and -poor compartments as visualized on stained adjacent sections. Thus, the distribution of dopamine D2 receptors conforms to the "striosomal" organization of the human caudate-putamen, a finding that suggests that this receptor subtype may mediate the influence of dopamine on distinct neurochemical compartments within the structure.
Aristotle revisited: the function of pyloric caeca in fish.
Authors: Buddington R K , Diamond J M
Published: May 2006

The function of the pyloric caeca of fish has been uncertain since their detailed description in 345 B.C. by Aristotle. He suggested three hypotheses about their function: "to store up the food," "putrify it up," and "concoct it" (i.e., storage, fermentation, and digestion). Our results for trout, cod, largemouth bass, and striped bass support the third but not the first or second of Aristotle's theories. In all four species, the caeca prove to be a major site of sugar, amino acid, and dipeptide uptake, contributing more uptake than the entire remaining alimentary tract in trout and cod. Caecal brush-border membranes contain hydrolytic enzymes. X-ray plates taken at various times after trout had ingested radioopaque marker, and observations of trout fed blue dye plus glass beads of graded sizes, show that caeca fill and empty of food with the same time course as proximal intestine. Thus, whereas the caeca of mammals and birds serve as fermentation chambers, fish caeca are an adaptation to increase gut surface area.
Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.
Authors: Vinogradov S N , Lugo S D , Mainwaring M G , Kapp O H , Crewe A V
Published: May 2006

The complete dissociation of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and III (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains II, III, and IV) and that peak II consisted of subunit M (16 kDa, chain I). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak Ib (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak Ib contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and III. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumbricus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were approximately equal to 20% smaller than those of the native hemoglobin.
Temperature dependence of the hydrophobic interaction in protein folding.
Authors: Baldwin R L
Published: May 2006

Accurate calorimetric data for the thermodynamics of transfer of six liquid hydrocarbons to water have been combined with solubility data to provide a model for the temperature dependence of the hydrophobic interaction in protein folding. The model applies at temperatures for which the change in heat capacity (delta Cp) is constant. The extrapolated value of the temperature (Ts) at which the entropy of transfer (delta S degrees) reaches zero is strikingly similar (Ts = 112.8 degrees C +/- 2.2 degrees C) for the six hydrocarbons. This finding provides an interpretation for the empirical relation discovered by Sturtevant: the ratio delta S degrees/delta Cp measured at 25 degrees C is constant for the transfer of nonpolar substances from nonaqueous media to water. Constancy of this ratio is equivalent to Ts = constant. When applied to protein folding, the hydrocarbon model gives estimates of the contributions of the hydrophobic interaction to the entropy and enthalpy changes on unfolding and, by difference, estimates of the residual contributions from other sources. The major share of the large enthalpy change observed on unfolding at high temperatures comes from the hydrophobic interaction. The hydrophobic interaction changes from being entropy-driven at 22 degrees C to being enthalpy-driven at 113 degrees C. Finally, the hydrocarbon model predicts that plots of the specific entropy change on unfolding versus temperature should nearly intersect close to 113 degrees C, as observed by Privalov.
Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae.
Authors: Makarow M , Nevalainen L T , Kääriäinen L
Published: May 2006

The nucleocapsid of vesicular stomatitis virus (VSV) was introduced into the cytoplasm of Saccharomyces cerevisiae by low pH-dependent fusion of the viral envelope with the spheroplast plasma membrane. This led to de novo synthesis of the three major structural proteins of the virus--the G, N, and M proteins--as shown by immunoprecipitation of [35S]methionine-labeled spheroplast lysates. In NaDodSO4/polyacrylamide gel electrophoresis, M and N proteins comigrated with those of the virion, whereas the yeast-made G protein migrated as two bands with apparent molecular sizes of 60 and 70 kDa. Both polypeptides appeared to be N-glycosylated, since only one polypeptide with the apparent molecular mass of approximately equal to 55 kDa was produced in the presence of tunicamycin. Phase separation into Triton X-114 suggested that the unglycosylated G protein was membrane bound. According to immunofluorescent surface staining of live spheroplasts, at least part of the G protein was transported to the plasma membrane. Spheroplasts expressing the VSV genes could be fused together by low pH to form polykaryons, indicating that G protein synthetized by yeast was fusogenic--i.e., biologically active.
Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
Authors: Fuerst T R , Niles E G , Studier F W , Moss B
Published: May 2006

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
Early regions of JC virus and BK virus induce distinct and tissue-specific tumors in transgenic mice.
Authors: Small J A , Khoury G , Jay G , Howley P M , Scangos G A
Published: May 2006

JC virus and BK virus are ubiquitous human viruses that share sequence and structural homology with simian virus 40. To characterize tissue-specific expression of these viruses and to establish model systems for the study of human viral-induced disease, transgenic mice containing early regions of each of the viruses were produced. The viral sequences induced tumors in a distinct and tissue-specific manner that was similar to their tissue tropism in humans. Ten JC virus-containing founder mice were produced, of which 5 survived to maturity. Four of them developed adrenal neuroblastomas, which metastasized to several other tissues. JC virus tumor-antigen RNA was detected at high levels in the tumor tissues and at low levels in the normal tissues of these mice. One of the three BK virus-containing mice was abnormally shaped and died at 2 weeks of age. The other two BK virus-containing mice developed primary hepatocellular carcinomas and renal tumors and died at 8-10 months of age. BK virus tumor-antigen RNA was expressed in tumor tissues of both mice. Since each of the viruses retained the general tissue tropism that it exhibits in humans, these data suggest that transgenic mice harboring human viruses will be useful as animal models for viral-induced diseases.
Altered I-J phenotype in E alpha transgenic mice.
Authors: Flood P M , Benoist C , Mathis D , Murphy D B
Published: May 2006

One of the more intriguing puzzles in immunology is the genetic basis for control of murine T-cell I-J determinants. Molecules bearing I-J determinants (I-J molecules) play a role in information trafficking among immunocompetent cells, probably serving as self-recognition molecules that channel regulatory factors to their appropriate target cells. Although it is clear that I-J polymorphism is influenced by the major histocompatibility complex (MHC), molecular genetic studies provide evidence that an MHC gene does not encode I-J molecules. A possible explanation for this paradox is that I-J molecules are a set of non-MHC-encoded T cell receptors that are directly or indirectly selected for by self-MHC products. One key to resolving the genetic and molecular basis for control of I-J determinants is the identification of the MHC gene(s) involved. Herein, data are presented which show that E alpha transgenic mice express an altered I-J phenotype, providing clear evidence that I region class II genes influence I-J polymorphism. Although further study is required to resolve how class II genes mediate this effect, this is a major piece to the I-J puzzle.
Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins.
Authors: Theurkauf W E , Baum H , Bo J , Wensink P C
Published: May 2006

We have determined the nucleotide sequences of all four Drosophila alpha-tubulin genes (alpha 1, alpha 2, alpha 3, and alpha 4). Two of the genes, alpha 1 and alpha 3, are constitutively expressed and code for proteins that are very similar to previously sequenced alpha-tubulins. They differ from each other by only two amino acid substitutions. These two genes also have blocks of homology between the noncoding leader regions of their transcription units. In contrast to these constitutive genes, the tissue-specific alpha 2 and alpha 4 genes code for tubulins with different structures. The alpha 2 mRNA is male-specific in adults and codes for a tubulin that differs from alpha 1 at 21 of the 450 residues. Six nonconservative substitutions are clustered within the 14 carboxyl-terminal amino acids, a region implicated in the regulation of microtubule assembly. The alpha 4 mRNA is maternal and is found only in ovarian nurse cells, eggs, and early embryos. It codes for the most highly divergent alpha-tubulin yet reported and differs from alpha 1 at 149 positions.
Structural homology of reaction centers from Rhodopseudomonas sphaeroides and Rhodopseudomonas viridis as determined by x-ray diffraction.
Authors: Allen J P , Feher G , Yeates T O , Rees D C , Deisenhofer J , Michel H , Huber R
Published: May 2006

Crystals of the reaction center (RC) from Rhodopseudomonas sphaeroides with the space group P2(1)2(1)2(1), have been studied by x-ray diffraction. The Patterson search (molecular replacement) technique was used to analyze the data, with the structure of the reaction center from Rhodopseudomonas viridis as a model system. A preliminary electron density map of the reaction center from R. sphaeroides has been obtained. Comparison of the structure of the RC from R. sphaeroides with that from R. viridis showed the following conserved features: five membrane-spanning helices in each of the L and M subunits, a single membrane-spanning helix in the H subunit, a 2-fold symmetry axis, and similar positions and orientations of the cofactors. Unlike the RCs from R. viridis, both quinones are retained in the RCs from R. sphaeroides. The secondary quinone is located near the position related by the 2-fold symmetry axis to the primary quinone.
An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila.
Authors: Wu M , Allis C D , Richman R , Cook R G , Gorovsky M A
Published: May 2006

An intervening sequence of 254 base pairs interrupts the coding region of the single gene for macronuclear histone H1 of the ciliated protozoan, Tetrahymena thermophila. The intervening sequence has splice junctions similar to those found in RNA polymerase II genes of other organisms. No obvious similarities are observed between this intron and the self-splicing intervening sequence of the Tetrahymena ribosomal gene. The derived amino acid sequence describes a small extremely basic H1 protein missing most of the central hydrophobic domain that is conserved in all other H1 proteins. Macronuclei divide amitotically, without chromosome condensation, suggesting the conserved globular domain of H1 plays a role in higher-order chromatin structure.
Genes for synapsin I, a neuronal phosphoprotein, map to conserved regions of human and murine X chromosomes.
Authors: Yang-Feng T L , DeGennaro L J , Francke U
Published: May 2006

Synapsin I is a neuron-specific phosphoprotein associated with the membranes of small synaptic vesicles. Its function is not entirely clear, but evidence points to a possible role in the regulation of neurotransmitter release. Its biosynthesis is under developmental control. Assignment of the human synapsin I gene to the X chromosome at band Xp11 was accomplished by in situ hybridization, using a rat cDNA probe. Southern blot analysis of DNAs from a panel of human-Chinese hamster somatic cell hybrids with defined regions of the human X chromosome confirmed the in situ mapping data. The mouse synapsin I gene was assigned to the X chromosome, proximal to band XD, by Southern blot analysis of Chinese hamster-mouse somatic cell hybrids with normal or rearranged mouse X chromosomes. In situ chromosomal hybridization experiments localized the mouse synapsin I gene more precisely to bands XA1----A4. These results add to the comparative gene map of mammalian species and support certain hypotheses regarding the evolutionary relationship between human and mouse X chromosomes. We hypothesize that the synapsin I gene could be mutated in human X-linked disorders with primary neuronal degeneration, such as the Rett syndrome.
A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.
Authors: Biron K K , Fyfe J A , Stanat S C , Leslie L K , Sorrell J B , Lambe C U , Coen D M
Published: May 2006

We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.
Structure and replication of the genome of the hepatitis delta virus.
Authors: Chen P J , Kalpana G , Goldberg J , Mason W , Werner B , Gerin J , Taylor J
Published: May 2006

The hepatitis delta virus can be found in the serum and liver of some hepatitis B virus patients. We now report that the RNA genome of serum-derived delta virus is single-stranded and circular. Livers of infected chimpanzees or woodchucks contained as many as 300,000 copies of genomic strand RNA per average cell, and at least some of this RNA had a circular conformation. Also present in the livers were RNA species complementary to the virion RNA. The genomic RNA was 5-22 times more abundant than this antigenomic strand. Some of the antigenomic RNA was complexed with genomic RNA, as evidenced by the fact that at least 34% of the antigenomic RNA was resistant to digestion with either RNase A in 0.3 M NaCl or S1 nuclease. Some of the antigenomic RNA was in a circular conformation. These and other findings showed that the structure and replication of hepatitis delta virus are in many ways similar to those of the previously described plant viroids, virusoids, and satellite RNAs.
Varicella zoster virus glycoprotein gpI is selectively phosphorylated by a virus-induced protein kinase.
Authors: Montalvo E A , Grose C
Published: May 2006

Varicella zoster virus glycoprotein I (VZV gpI; Mr 98,000) was phosphorylated in virus-infected human cell monolayers, while two other major VZV glycoproteins (gpII and gpIII) were not similarly modified. Phosphorylation of VZV gpI was not blocked by inhibitors of glycosylation, nor were the phosphoryl groups enzymatically removed by endoglycosidases. Phosphoamino acid analysis revealed the presence of phosphoserine and phosphothreonine residues on the polypeptide backbone. The selective nature of the phosphorylation event was further demonstrated in vitro by a protein kinase (Mr 50,000), which was present in virus-infected cells but absent from uninfected cells or purified virions. The enzyme catalyzed the transfer of 32Pi from [gamma-32P]ATP to gpI but not to gpII and gpIII. Like VZV gpI, this virus-induced protein kinase was also a constituent of the plasma membrane of live VZV-infected cells.
Fluorescence polarization decay of tyrosine in lima bean trypsin inhibitor.
Authors: Nordlund T M , Liu X Y , Sommer J H
Published: May 2006

The fluorescence of lima bean trypsin inhibitor is due to a single tyrosine residue at position 69. The lifetime of this tyrosine fluorescence is 620 +/- 50 ps (mean +/- SD) and is little affected by addition of 0.88 M citrate, an efficient quencher of tyrosine fluorescence. The steady-state emission intensity is also only weakly reduced by the quencher. The tyrosine is thus not accessible to the citrate and is probably located in the interior of the protein. The high pK of the tyrosine supports this conclusion. The fluorescence anisotropy decay of the inhibitor's tyrosine can be fitted to a double exponential form, with time constants of about 40 ps and greater than or equal to 3 ns. The anisotropy at time zero is 0.19 +/- 0.015 (mean +/- SD), the same as for N-acetyl-L-tyrosinamide in viscous glycerol solution. The nanosecond component of the decay is consistent with rotation of the entire protein molecule. The 40-ps component demonstrates that the tyrosine has considerable freedom to move independently of the protein as a whole. This rotational correlation time is approximately what is observed for free tyrosine in aqueous solution. Since the polypeptide chain near tyrosine-69 is anchored by several disulfide bonds, the data argue that this interior portion of the protein consists of a rigid, immobile backbone embedded in fluid, mobile amino acid side chains.
Nucleotide binding by a 24-residue peptide from the RecA protein of Escherichia coli.
Authors: Knight K L , McEntee K
Published: May 2006

We have recently demonstrated that two ATP analog affinity labels, 8-azidoadenosine 5'-triphosphate (N3ATP) and 5'-p-fluorosulfonylbenzoyladenosine (5'FSBA), covalently modify RecA protein of Escherichia coli at a specific tyrosine residue (Tyr-264) located within a 24-residue tryptic peptide (T-31) spanning residues 257-280. Here we show that N3ATP efficiently modifies purified peptide T-31 and show that the interaction is specific by the following criteria: photolabeling of peptide T-31 is saturable with respect to the N3ATP concentration; photolabeling is competitive with ATP and adenosine but not with adenine, UTP, or TTP; and other peptides derived from RecA protein were poor substrates for photolabeling except for one fragment that showed a nonspecific interaction with the photoaffinity analog. Analysis of N3ATP-modified T-31 shows that the photolabel attaches to more than one site within the peptide. These data argue that peptide T-31 contains some sites of contact for adenine and ribose moieties of ATP when it is bound to RecA protein.
Multisite phosphorylation of the alpha subunit of transducin by the insulin receptor kinase and protein kinase C.
Authors: Zick Y , Sagi-Eisenberg R , Pines M , Gierschik P , Spiegel A M
Published: May 2006

The GDP-bound alpha subunit of transducin, but not the guanosine 5'-[gamma-thio]triphosphate-bound one, undergoes phosphorylation on tyrosine residues by the insulin receptor kinase and on serine residues by protein kinase C. Holotransducin is poorly phosphorylated by the insulin receptor kinase and is not phosphorylated by protein kinase C. Neither holotransducin nor any of its subunits were phosphorylated by the cAMP-dependent protein kinase. That a given subunit of transducin undergoes multisite phosphorylation depending on the type of nucleotide bound to it or the nature of the kinase suggests that hormone-dependent phosphorylation could provide a versatile mode for regulation of guanine nucleotide-binding protein (G protein) function. In particular, the findings that certain G proteins serve as substrates for both the insulin receptor kinase and protein kinase C implicate G proteins in playing a key role in mediating the action of insulin and ligands that act to activate protein kinase C.
Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species.
Authors: Charbonneau H , Beier N , Walsh K A , Beavo J A
Published: May 2006

Partial amino acid sequences have been determined for the Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase from bovine brain and the cGMP-stimulated cyclic nucleotide phosphodiesterase from bovine heart. Examination of these sequences for homologous segments and comparison with protein sequences derived from the nucleotide sequences of the yeast PDE2 gene and the Drosophila dunce+ gene [Chen, C.-N., Denome, S. & Davis, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 9313-9317; Sass, P., Field, J., Nikawa, J., Toda, T. & Wigler, M. (1986) Proc. Natl. Acad. Sci. USA 83, 9303-9307] reveal a 200- to 270-residue segment in each that is homologous to the others. The molecular masses of the four proteins vary from 40 kDa to 105 kDa, and the structural resemblance appears to be constrained to a single segment of each protein. These related segments are proposed to comprise the catalytic domains in this set of enzymes. The lack of absolute sequence identity between the two bovine enzymes shows that they are unique gene products that are not produced by alternative processing of a larger protein or of a single mRNA precursor. The data also strongly support the conclusion that the dunce+ gene locus of Drosophila and the PDE2 gene locus in yeast code for structural genes of cyclic nucleotide phosphodiesterases.
Functional expression of a human Na+/H+ antiporter gene transfected into antiporter-deficient mouse L cells.
Authors: Franchi A , Perucca-Lostanlen D , Pouyssegur J
Published: May 2006

To clone the gene for the human Na+/H+ antiporter, we first constructed a stable mouse LTK- cell line (LAP1) lacking Na+/H+ antiport activity. Second, we devised a selective technique based on acid killing that specifically sorts out cells expressing low levels of Na+/H+ antiport activity from a population of antiporter-deficient cells (AP-). LAP1 cells (TK- and AP-) were cotransformed with human genomic DNA and the thymidine kinase (TK) gene. TK+ transformants, first selected, were submitted to acid loading. The rare transformants that survived (frequency, 2-8 X 10(-7) expressed Na+/H+ antiport activity (AP+). We found that: transformation with mouse LAP1 DNA did not give rise to AP+ transformants; transformation of LAP1 cells with DNA from an altered Na+/H+ antiporter hamster variant led to AP+ transformants expressing the altered Na+/H+ antiporter of the DNA donor; human repeated sequences were present in all primary, secondary, and tertiary mouse AP+ transformants; six identical EcoRI human DNA fragments (55 kilobase pairs of the human genome) cosegregated with the Na+/H+ antiport activity in secondary and tertiary transformants. These results strongly suggest that we have stably expressed the structural gene for the human Na+/H+ antiporter in mouse cells.
Multiple controls of oxidative metabolism in living tissues as studied by phosphorus magnetic resonance.
Authors: Chance B , Leigh J S , Kent J , McCully K , Nioka S , Clark B J , Maris J M , Graham T
Published: May 2006

Three types of metabolic control of oxidative metabolism are observed in the various tissues that have been studied by phosphorous magnetic resonance spectroscopy. The principal control of oxidative metabolism in skeletal muscle is by ADP (or Pi/phosphocreatine). This conclusion is based upon studies of arm muscles of humans during steady-state exercise. A work-cost (Vm vs. Pi/phosphocreatine) relationship follows a Michaelis-Menten rectangular hyperbola, where Km values from 0.5 to 0.6 and Vmax values from 50 to 200 (at nearly constant pH) are found in linearized plots of the equation V/Vmax = 1/(1 + 0.6 phosphocreatine/Pi) where V is work level (which is equal to the velocity of the enzymatic reaction) and Vmax is the maximal work capacity that is a measure of the enzyme activity (E) of oxidative metabolism. Adaptation to exercise enhances the slope of the work-cost relationship and causes large changes in Vmax or E. A second metabolic control may enhance the slope of the work-cost relationship but not Vmax. For example, the initiation of exercise can lead to an improved characteristic that can be explained by 2-fold increased substrate delivery, for example, increased oxygen delivery by microcirculatory control. Cardiac tissue of the adult dog affords an example of optimal endurance performance adaptation and exhibits the steepest work-cost relationship observed and is attributed to a coordinated control of substrate delivery that may involve Ca2+ and inorganic phosphate control of NADH; control of O2 delivery may also be involved. The calculated work-cost relationship is similar to that observed in the beagle heart. The theoretical curve illustrates that the liability of multiple controls is a sharp break point in metabolic control at the end of the multiple control range--a possible cause of instability of cardiac performance at high V/Vmax.
Behavioral facilitation of reproduction in sexual and unisexual whiptail lizards.
Authors: Crews D , Grassman M , Lindzey J
Published: May 2006

All-female, parthenogenetic species afford a unique test of hypotheses regarding the nature and evolution of sexuality. Mating behavior accomplishes the transfer of gametes and stimulates the coordination of reproductive activity of the male and female. Cnemidophorus uniparens, a parthenogenetic species, is believed to have resulted from the hybridization of two extant gonochoristic species, Cnemidophorus inornatus and Cnemidophorus gularis. C. uniparens regularly and reliably perform behaviors identical in form to those performed during mating by male C. inornatus. We have determined experimentally that individuals of the parthenogenetic species demonstrating male-like pseudosexual behavior also share a similarity in function with males of the sexually reproducing species. The number of female C. inornatus ovulating increases, and the latency to ovulation decreases, if a sexually active conspecific male is present. A similar facilitatory effect on ovarian recrudescence occurs in the all-female C. uniparens in the presence of a male-like individual. These results show that behavioral facilitation of ovarian recrudescence is important in sexual and unisexual species. This may represent a potent selection pressure favoring the maintenance of male-typical behaviors, thus accounting for the display of behavioral traits usually associated with males in unisexual species of hybrid origin.
Nucleotide sequence, tissue-specific expression, and chromosome location of human carbonic anhydrase III: the human CAIII gene is located on the same chromosome as the closely linked CAI and CAII genes.
Authors: Wade R , Gunning P , Eddy R , Shows T , Kedes L
Published: May 2006

The carbonic anhydrases (CA) are a class of metalloenzymes that catalyze the reversible hydration of carbon dioxide. The genes for the carbonic anhydrase isozymes are members of a multigene family that are differentially expressed in a number of cell types. We have isolated a full-length representative of a CAIII mRNA transcript from an adult human muscle cDNA library, and we present the complete nucleotide sequence of this cDNA clone. RNA blots demonstrate that CAIII messages can be detected in a variety of cell types but that high-level expression is limited to human fetal and adult skeletal muscle and to rodent slow skeletal muscle and liver. In addition, we have used a panel of human-mouse cell hybrids to localize the human CAIII gene to chromosome 8. Previous reports have established the CAI and CAII isozyme genes to be closely linked on chromosome 8, and the assignment of the CAIII gene to the same chromosome raises the possibility that these genes may all be linked at a single complex locus.
Adenoviruses of subgenera B, C, D, and E modulate cell-surface expression of major histocompatibility complex class I antigens.
Authors: Pääbo S , Nilsson T , Peterson P A
Published: May 2006

The immune defense against viral infections involves cytotoxic T lymphocytes that recognize viral products in the context of class I major histocompatibility complex (MHC) antigens. To evade such immune surveillance viruses may have evolved various strategies to manipulate the expression of class I antigens. Adenovirus 2 manufactures an early glycoprotein, E19, that binds to nascent class I antigens in the endoplasmic reticulum and impedes their transport to the cell surface. We now show that adenoviruses typical of all viral subgenera except the highly oncogenic subgenus A dramatically reduce the cell-surface expression of class I antigens. It has been shown that subgenus A viruses abolish class I antigen expression in transformed cells by reducing mRNA levels. Thus, all adenoviruses can modulate the cell-surface expression of class I antigens.
Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses.
Authors: Gendelman H E , Phelps W , Feigenbaum L , Ostrove J M , Adachi A , Howley P M , Khoury G , Ginsberg H S , Martin M A
Published: May 2006

To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.
On the mechanism of the chemical and enzymic oxygenations of alpha-oxyprotohemin IX to Fe.biliverdin IX alpha.
Authors: Sano S , Sano T , Morishima I , Shiro Y , Maeda Y
Published: May 2006

alpha-Oxyprotohemin IX, an early intermediate in heme catabolism, was synthesized and its autoxidation to biliverdin IX alpha was studied. In anaerobic aqueous pyridine, alpha-oxyprotohemin (hexacoordinated) underwent autoreduction to yield an Fe(II) alpha-oxyprotoporphyrin pi-neutral radical bis(pyridine) complex, which reacted with an equimolar amount of dioxygen to give pyridine.verdohemochrome IX alpha and CO in 75-80% yield via an intermediate with an absorption maximum at 893 nm. Verdohemochrome IX alpha did not react with further dioxygen. Reconstituted apomyoglobin.alpha-oxyprotohemin IX complex (pentacoordinated) reacted with an equimolar amount of dioxygen to form an Fe(II) oxyporphyrin pi-neutral radical intermediate, which rearranged to a green compound (lambda max 660 and 704 nm) with elision of CO. The green product, which is probably an apomyoglobin.verdoheme pi-radical complex, reacted with another equimolar amount of dioxygen to give Fe(III).biliverdin IX alpha. Demetallation of this gave biliverdin IX alpha in overall yield of 70-75%. These results indicate that the sequence of oxyheme autoxidation in the presence of apomyoglobin is alpha-oxyprotoheme IX O2----CO----verdohemochrome IX alpha pi-radical O2----Fe(III).biliverdin IX alpha. A similar mechanism may prevail in vivo. The hexa- and pentacoordinated Fe(II) pi-radical form of the oxyporphyrin is crucial in triggering the autoxidation of the complex to verdohemochrome IX alpha. Further oxygenation of verdohemochrome IX alpha to Fe(III).biliverdin IX alpha occurred only in the pentacoordinated apomyoglobin.verdoheme Fe(II) complex.
Reconstruction of the dynamics of the climatic system from time-series data
Authors: Nicolis C. , Nicolis G.
Published: May 2006

The oxygen isotope record of the last million years, as provided by a deep sea core sediment, is analyzed by a method recently developed in the theory of dynamical systems. The analysis suggests that climatic variability is the manifestation of a chaotic dynamics described by an attractor of fractal dimensionality. A quantitative measure of the limited predictability of the climatic system is provided by the evaluation of the time-correlation function and the largest positive Lyapounov exponent of the system.
Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.
Authors: Tapprich W E , Hill W E
Published: May 2006

A nine-base DNA oligomer [d(GTATCTAAT)] was used to probe the accessibility and function of bases in the region 787-795 of Escherichia coli 16S rRNA. Hybridization of the cDNA [d(GTATCTAAT)] to 16S rRNA in situ was carried out by binding the probe to intact 30S ribosomal subunits. Nitrocellulose filter binding showed that cDNA hybridization saturated with increasing probe concentration, suggesting that the probe was binding to a discrete site or sites. RNase H digestion of the rRNA under the DNA . rRNA hybrid and sequencing of the resultant RNA fragments verified that the cDNA probe bound specifically to the 787-795 region. Hybridization experiments using the cDNA probe showed that bases in the 787-795 region of 16S rRNA are exposed on the surface of 30S subunits. The functional role of bases 787-795 was then tested by assaying various ribosomal activities with the cDNA in place. Results of these functional assays demonstrate that this 16S rRNA region is directly involved in the association of 30S and 50S subunits.
Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli.
Authors: Sung W L , Yao F L , Zahab D M , Narang S A
Published: May 2006

Enhanced accumulation of human proinsulin synthesized in Escherichia coli has been achieved by inserting a short leader of homooligopeptide at the amino end of proinsulin. Out of 20 amino acid oligomers studied, (Ala)6, (Asn)6, (Cys)7, (Gln)7, (His)6, (Ser)6, and (Thr)6 leaders were the most effective, with the yield of proinsulin ranging between 6% and 26% of the total bacterial protein. These constructions were made by inserting a synthetic oligodeoxyribonucleotide duplex, coding for a small homooligopeptide, between a synthetic proinsulin gene and an eight-codon beta-galactosidase gene residue in vector pUC8. Cyanogen bromide cleavage of the 102 amino acid fused polypeptide yielded a species identical to authentic proinsulin, as judged by NaDodSO4/PAGE and radioimmunoassay.
Distinctive role of histidine-16 of the B beta chain of fibrinogen in the end-to-end association of fibrin.
Authors: Shimizu A , Saito Y , Inada Y
Published: May 2006

Photooxidation of fibrinogen reduced the batroxobin-induced fibrin polymerization. The fibrin fragment des-AB N-DSK, which contains the binding sites termed A and B, lost the ability to bind to the site termed a in fibrinogen-Sepharose upon the oxidation of histidine-16 in the B beta chain of fibrinogen [Shimizu, A., Saito, Y., Matsushima, A. & Inada, Y. (1983) J. Biol. Chem. 258, 7915-7917]. Some of the fragments, which became unable to bind to fibrinogen-Sepharose due to the destruction of site A, however, retained the ability to bind to D-dimer-Sepharose, which contains both sites a and b. This shows that histidine-16 of the B beta chain of fibrinogen is essential for site A but may not be essential for site B. It is of interest that histidine-16 of the B beta chain, which is only one residue away from the thrombin-susceptible bond, makes a part of the site A for the end-to-end association created by the release of fibrinopeptide A.
Ganglioside GM3: an acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiation of human myeloid and monocytoid leukemic cell lines HL-60 and U937.
Authors: Nojiri H , Takaku F , Terui Y , Miura Y , Saito M
Published: May 2006

When human myeloid and monocytoid leukemic cell lines HL-60 and U937, respectively, were treated with an exogenous sialoglycosphingolipid, ganglioside GM3, in serum-free medium, cell growth was markedly inhibited, and their morphological maturation along a monocytic lineage was observed. In addition to a significant increase in phagocytic and nonspecific esterase activities, marked increase of monocyte-specific surface antigens detectable with monoclonal antibodies such as OKM1 and OKM5 was observed in GM3-fed cells. Other sialoglycosphingolipids with the carbohydrate structure belonging to ganglio-series oligosaccharide, ganglioside GM1 and a brain ganglioside mixture, had no effect on the cell differentiation, showing instead stimulatory actions on the growth of these cell lines. We recently demonstrated that the ganglio-series ganglioside GM3 characteristically increased during macrophage-like cell differentiation of these cell lines. The present results indicate that ganglioside molecular species that specifically increase during monocytic cell differentiation of human myeloid and monocytoid leukemic cell lines may play, in turn, an important role in the differentiation-induction of these cell lines along a monocytic cell lineage.
Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3.
Authors: Kindler V , Thorens B , de Kossodo S , Allet B , Eliason J F , Thatcher D , Farber N , Vassalli P
Published: May 2006

Mouse interleukin 3 (IL-3) cDNA was cloned into a plasmid construction, allowing the synthesis of very high quantities of IL-3 in Escherichia coli. The recombinant (r) IL-3, purified to homogeneity, was active in vitro on the proliferation and differentiation of various hematopoietic progenitor cells at 1 pM. To maintain detectable blood levels of IL-3, osmotic pumps containing rIL-3 or control solutions were placed under the skin of normal and irradiated C3H/HeJ and (BALB X B10) F1 mice. The effect of IL-3 on hematopoietic progenitor cell numbers in spleen and bone marrow was evaluated 3 and 7 days later by using an in vitro clonal assay. The results demonstrated the following: (i) Doses of IL-3 infused at the rate of 2.5-5 ng per g of body weight per hr were sufficient to increase the numbers of hematopoietic progenitors in normal mice by at least 2-fold within 3 days. (ii) In mice with progenitor cell levels depressed by sublethal irradiation, 7-day treatment with IL-3 resulted in a 10-fold increase to near normal levels. (iii) The erythroid and myeloid lineages appeared to be enhanced to the same extent. (iv) Enhancement of hematopoiesis occurred primarily in spleen, but hematopoietic foci were also evident in the liver; in contrast, total cell and progenitor cell numbers were decreased in the bone marrow.
Human small-cell lung cancers show amplification and expression of the N-myc gene.
Authors: Nau M M , Brooks B J , Carney D N , Gazdar A F , Battey J F , Sausville E A , Minna J D
Published: May 2006

We have found that 6 of 31 independently derived human small-cell lung cancer (SCLC) cell lines have 5- to 170-fold amplified N-myc gene sequences. The amplification is seen with probes from two separate exons of N-myc, which are homologous to either the second or the third exon of the c-myc gene. Amplified N-myc sequences were found in a tumor cell line started prior to chemotherapy, in SCLC tumor samples harvested directly from tumor metastases at autopsy, and from a resected primary lung cancer. Several N-myc-amplified tumor cell lines also exhibited N-myc hybridizing fragments not in the germ-line position. In one patient's tumor, an additional amplified N-myc DNA fragment was observed and this fragment was heterogenously distributed in liver metastases. In contrast to SCLC with neuroendocrine properties, no non-small-cell lung cancer lines examined were found to have N-myc amplification. Fragments encoding two N-myc exons also detect increased amounts of a 3.1-kilobase N-myc mRNA in N-myc-amplified SCLC lines and in one cell line that does not show N-myc gene amplification. Both DNA and RNA hybridization experiments show that in any one SCLC cell line, only one myc-related gene is amplified and expressed. We conclude that N-myc amplification is both common and potentially significant in the tumorigenesis or tumor progression of SCLC.