bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study.
Authors: Cortés R , Villar M J , Verhofstad A , Hökfelt T
Published: May 2006

We have used in situ hybridization and immunohistochemistry to study the expression of galanin mRNA and galanin-like immunoreactivity after decortication and lesions of the ventral hippocampus. After decortication the levels of both galanin mRNA and galanin-like immunoreactivity were increased in the dorsal raphe nucleus. In addition, in decorticated rats, but not in controls, galanin mRNA could be seen in dorsal and ventral nuclei of the thalamus and in the remaining parts of the cortex. Increases in galanin mRNA and galanin-like immunoreactivity were also observed in the septum-vertical diagonal band after electrocoagulation lesions of the ventral hippocampus. In contrast, no changes were found after ibotenic acid lesions of the same hippocampal area. These results suggest that increases in the expression of galanin occur in certain neuron populations after direct lesion of their axons and/or terminal fields.
Chemical carcinogenesis: too many rodent carcinogens.
Authors: Ames B N , Gold L S
Published: May 2006

The administration of chemicals at the maximum tolerated dose (MTD) in standard animal cancer tests is postulated to increase cell division (mitogenesis), which in turn increases rates of mutagenesis and thus carcinogenesis. The animal data are consistent with this mechanism, because a high proportion--about half--of all chemicals tested (whether natural or synthetic) are indeed rodent carcinogens. We conclude that at the low doses of most human exposures, where cell killing does not occur, the hazards to humans of rodent carcinogens may be much lower than is commonly assumed.
TPR-MET oncogenic rearrangement: detection by polymerase chain reaction amplification of the transcript and expression in human tumor cell lines.
Authors: Soman N R , Wogan G N , Rhim J S
Published: May 2006

Activation of the MET protooncogene by a rearrangement involving the fusion of TPR and MET specific gene sequences has been observed in a human osteosarcoma cell line (HOS) treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No information has been available about the possible occurrence of this rearrangement in human tumors. To facilitate rapid screening of human cell lines and tumor samples for this specific gene rearrangement, we developed a sensitive detection method based on polymerase chain reaction (PCR) amplification of TPR-MET mRNA. cDNA was generated from cellular transcripts by using one of the PCR primers, which was then used as a template for PCR amplification of a 205-base-pair region carrying the breakpoint. An end-labeled internal probe was hybridized in solution to an aliquot of the PCR product for detecting amplification. Cells could be directly screened by the assay without prior isolation of RNA. A 205-base-pair DNA fragment characteristic of the TPR-MET rearrangement was detected in cell lines previously known to contain this altered sequence. The rearrangement was also detected at very low levels in the parental (nontransformed) cell line, HOS TE-85. A preliminary survey of cell lines derived from a variety of human tumors indicates that TPR-MET rearrangement occurred and was expressed at very low frequencies by cells from 7 of 14 tumors of nonhematopoietic origin.
Isolation and initial characterization of a specific premessenger ribonucleoprotein particle.
Authors: Wurtz T , Lönnroth A , Ovchinnikov L , Skoglund U , Daneholt B
Published: May 2006

A specific type of premessenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules, has been isolated from heterogeneous nuclear RNP (hnRNP) in the salivary glands of the dipteran Chironomus tentans. A BR granule contains a single 75S RNA molecule coding for a large secretory protein (Sp1). The isolation procedure is based on the abundance and exceptional size of the BR granules: in EDTA-containing sucrose gradients they sediment as a sharp 300S peak ahead of the remainder of the hnRNP population. The isolated BR granules were identified on the basis of both ultrastructural and biochemical criteria: large spherical particles that contain 75S RNA and BR sequences. A three-dimensional reconstruction of isolated particles by electron microscope tomography further supported the identification of the isolated particles as BR granules. In contrast to the entire hnRNP population, the BR granules exhibited a sharp peak in CsCl gradients with a buoyant density of 1.45 g/cm3. This result indicates that a BR granule consists of 40% RNA and 60% protein by weight, corresponding to a 75S RNA molecule of 12 megadaltons and a total protein content of 18 megadaltons, or about 500 average-sized protein molecules.
Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons.
Authors: Mochida S , Poulain B , Eisel U , Binz T , Kurazono H , Niemann H , Tauc L
Published: May 2006

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons. If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context. To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations. Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known. Both toxins block neurotransmitter release at nanomolar intracellular concentrations. These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced. Their active sites are located (or partly located) on the light chain. mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ.
Evidence from electron micrographs that icosahedral quasicrystals are icosahedral twins of cubic crystals.
Authors: Pauling L
Published: May 2006

An analysis of electron micrographs of Al5Mn quasicrystals obtained by rapidly cooling a molten alloy with composition Al17Mn and removing the Al matrix by electrosolution, revealing aggregates of 20 microcrystals at the corners of a pentagonal dodecahedron, supports the proposal that these microcrystals are cubic crystals twinned about an icosahedral seed, with each cubic microcrystal sharing a threefold axis and three symmetry planes with the seed.
Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity.
Authors: Satoh T , Endo M , Nakafuku M , Akiyama T , Yamamoto T , Kaziro Y
Published: May 2006

The ras gene product (p21) is a GTP-binding protein and has been thought to transduce signals regulating proliferation or differentiation of cells. Like other GTP-binding proteins, p21.GTP is an active conformation, which can transduce the signals downstream, whereas p21.GDP is an inactive one. Recently, we have shown that p21.GTP levels increased in cells treated with fetal bovine serum or platelet-derived growth factor to initiate DNA synthesis. In this paper, we report that epidermal growth factor can also increase the amounts of p21.GTP in the cells. Effects of epidermal growth factor and platelet-derived growth factor are not additive. In contrast, mutant [Val12]p21, which has transforming activity, responded neither to platelet-derived growth factor nor to epidermal growth factor. We also found that the ratio of p21.GTP to p21.GDP increased 3- to 4-fold in transformants carrying activated erbB-2/neu or v-src oncogenes. These results strongly suggest an important role of p21 in transduction of signals for both normal proliferation and malignant transformation through growth factor receptors with tyrosine kinase activity or related oncogene products.
Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide.
Authors: Price D J , Gunsalus J R , Avruch J
Published: May 2006

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated 32P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa 32P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified 32P-labeled H4 hepatoma insulin-stimulated S6 kinase. This antiserum also specifically precipitates insulin-stimulated S6 kinase activity directly from cytosolic extracts of H4 cells. Immune complexes prepared from the cytosol of 32P-labeled H4 cells contain several 32P-labeled polypeptides; only a 70-kDA 32P-labeled peptide, however, is specifically displaced by preadsorption of the antiserum with nonradioactive rat liver S6 kinase. Insulin treatment increases the 32P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels; 32P-labeled serine, some 32P-labeled threonine, but no 32P-labeled tyrosine are detected after partial acid hydrolysis. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from 32P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. Autophosphorylation of rat liver S6 kinase in vitro does not modify S6 kinase activity. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. The phosphatase 2A-deactivated 70-kDa S6 kinase is neither reactivated nor phosphorylated by partially purified insulin-stimulated microtubule-associated protein 2 kinase, in experiments where Xenopus S6 kinase II undergoes phosphorylation and partial reactivation. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.
HeT DNA: a family of mosaic repeated sequences specific for heterochromatin in Drosophila melanogaster.
Authors: Valgeirsdóttir K , Traverse K L , Pardue M L
Published: May 2006

HeT DNA is a complex family of repeated DNA found only in pericentric and telomeric heterochromatin. In contrast to other DNA families that have been specifically associated with heterochromatin, HeT DNA is not principally a family of tandemly repeated elements. Much of the HeT DNA family appears to be a mosaic of several different classes of large sequence elements arranged in a scrambled array; however, some elements of the family can be found in tandem repeats. In spite of the variable order of the different elements in HeT DNA, the sequence homology between different members of each class of element is extremely high, suggesting that the members are evolving in a concerted fashion. Sequence analysis suggests that some elements in the HeT family may make up a novel family of heterochromatin-specific transposable elements and that the mosaic organization of the elements may be produced by retroposition and other mechanisms involved in the transposition of mobile elements. We suggest that such mechanisms may be a general feature for the maintenance of chromosome structure.
N-acetylaspartylglutamate: a transmitter candidate for the retinohypothalamic tract.
Authors: Moffett J R , Williamson L , Palkovits M , Namboodiri M A
Published: May 2006

The retinohypothalamic tract is the neural pathway mediating the photic entrainment of circadian rhythms in mammals. Important targets for these retinal fibers are the suprachiasmatic nuclei (SCN) of the hypothalamus, which are thought to be primary sites for the biological clock. The neurotransmitters that operate in this projection system have not yet been determined. Immunohistochemistry and radioimmunoassay performed with affinity-purified antibodies to N-acetylaspartylglutamate (NAAG) demonstrate that this neuron-specific dipeptide, which may act as an excitatory neurotransmitter, is localized extensively in the retinohypothalamic tract and its target zones, including the SCN. Optic nerve transections resulted in significant reductions in NAAG immunoreactivity in the optic chiasm and SCN. Analysis of NAAG concentrations in micropunches of SCN, by means of radioimmunoassay, showed approximately 50% reductions in NAAG levels. These results suggest that this peptide may act as one of the neurotransmitters involved in retinohypothalamic communication and circadian rhythm entrainment.
Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation.
Authors: Sternberg N , Coulby J
Published: May 2006

The packaging of bacteriophage PI DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full. We have previously shown that pac is a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of pac is methylation sensitive. Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not. Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase. Comparison of that uncut pac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated. In contrast, virion DNA containing a cut pac site was not undermethylated. We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle.
Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.
Authors: Partono S , Lewin A S
Published: May 2006

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.
Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse.
Authors: Lin J W , Sugimori M , Llinás R R , McGuinness T L , Greengard P
Published: May 2006

The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.
Transforming growth factor beta and noncytopathic mechanisms of immunodeficiency in human immunodeficiency virus infection.
Authors: Kekow J , Wachsman W , McCutchan J A , Cronin M , Carson D A , Lotz M
Published: May 2006

This study examines the contribution of transforming growth factor beta (TGF beta), one of the most potent endogenous immunosuppressive factors, to the development of immunodeficiency in human immunodeficiency virus (HIV) infection. Increased titers of TGF beta were found in supernatants of peripheral blood mononuclear cells (PBMCs) from HIV-infected donors as compared to uninfected controls (P less than 0.001). This correlated closely with defective responses of CD4+ lymphocytes to the recall antigens tuberculin purified protein derivative or tetanus toxoid. The addition of TGF beta-neutralizing antibody to PBMCs partially restored these defective T-cell responses. Furthermore, purified TGF beta or HIV+ PBMC culture supernatants preferentially inhibited proliferation of CD4+ lymphocytes as compared to CD8+ cells. The increased expression of the TGF beta protein was associated with increased TGF beta mRNA as determined by a polymerase chain reaction assay. This increase in TGF beta protein and mRNA was due to a selective upregulation of the TGF beta 1 isoform. These results indicate that overexpression of TGF beta 1 occurs in HIV-infected individuals and that this cytokine can contribute to impaired immune functions and to depletion of CD4+ T lymphocytes.
A principle for the formation of the spatial structure of cortical feature maps.
Authors: Obermayer K , Ritter H , Schulten K
Published: May 2006

Orientation-selective cells in the striate cortex of higher animals are organized as a hierarchical topographic map of two stimulus features: (i) position in visual space and (ii) orientation. We show that the observed structure of the topographic map can arise from a principle of continuous mapping. For the realization of this principle we use a mathematical model that can be interpreted as an adaptive process changing a set of synaptic weights, or synaptic connection strengths, between two layers of cells. The patterns of orientation preference and selectivity generated by the model are similar to the patterns seen in the visual cortex of macaque monkey and cat and correspond to a neural projection that maps a more than two-dimensional feature space onto a two-dimensional cortical surface under the constraint that shape and position of the receptive fields of the neurons very smoothly over the cortical surface.
Functional analysis of protein N-myristoylation: metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution.
Authors: Johnson D R , Cox A D , Solski P A , Devadas B , Adams S P , Leimgruber R M , Heuckeroth R O , Buss J E , Gordon J I
Published: May 2006

Covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97) and offer the opportunity to explore structure/function relationships of myristate in N-myristoyl proteins. We have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC3H1), (ii) a rat fibroblast cell that produces p60v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys----Ser-186 mutation. This latter mutation prevents isoprenylation and palmitoylation of ras. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins (i.e., protein-sequence-specific incorporation). In addition, proteins were identified that underwent redistribution from membrane to soluble fractions after incorporating one but not another analog (analog-specific redistribution). Comparable studies using the model RaSV-ras chimeric protein also demonstrated analog-specific differences in incorporation, varying from approximately 25% of the total RaSV-ras chimeric protein with 5-octyloxypentanoate to greater than 50% with 12-methoxydodecanoate. Modification by this latter compound was so extensive that the amount of membrane-associated N-myristoylated protein was decreased. Incorporation of each of the analogs caused a dramatic redistribution to the soluble fraction, comparable to that seen when myristoylation was completely blocked by mutating the protein's site of myristate attachment (glycine) to an alanine residue. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value.
A dorso-ventral asymmetry in the embryonic retina defined by protein conformation.
Authors: McCaffery P , Neve R L , Dräger U C
Published: May 2006

In a search for determinants of retinotopic specification we previously identified an antigen in the dorsal embryonic retina as a protein called the 68-kDa laminin receptor. A dorso-ventral asymmetry in a laminin receptor seemed consistent with the known responsiveness of embryonic optic axons to laminin, but there were three peculiar points. (i) The molecular mass of this presumed laminin receptor in immunoblots is not 68 kDa but 43 kDa, and the molecular mass of the protein deduced from the mRNA is only 33 kDa. (ii) The antigen does not have the localization expected of a receptor for the extracellular matrix: the antibodies label mainly a granular cytoplasmic antigen in dorsal retina; an additional sparse cell-surface antigen present on a few cells does not show a dorso-ventral asymmetry. (iii) Despite the pronounced dorso-ventral difference seen immunohistochemically, in immunoblots the 43-kDa protein (p40) is evenly distributed throughout the retina. Here we show that (i) native p40 and in vitro-translated gene product are indistinguishable and their anomalous migration in denaturing gels probably is due to low pI; (ii) p40 is bound in a Mg2(+)-dependent manner to large cytoplasmic complexes that appear to include ribosomes; and (iii) there is a labile conformational difference in p40 between dorsal and ventral retina: dorsally it is more accessible to proteolysis, suggesting a more open conformation. In conjunction with the recent hypothesis that p40 constitutes a translation initiation factor (D. Auth and G. Brawerman, personal communication), these observations point to a dorso-ventral asymmetry in some aspect of protein translation, which in turn may set up differences in recognition factors on retinal growth cones.
Streamlined approach to creating yeast artificial chromosome libraries from specialized cell sources.
Authors: Feingold J M , Ogden S D , Denny C T
Published: May 2006

The study of tumor-specific chromosomal abnormalities has been severely impeded by an inability to link cytogenetic to molecular data. Restriction fragment length polymorphism mapping of any particular chromosomal rearrangement to the resolution limit of genetic methodology generates sets of probes that frequently are still too widely spaced to render the rearrangement breakpoints accessible to molecular isolation. The stable propagation of genomic fragments of up to one million base pairs in size as yeast artificial chromosomes (YACs) represents an important development in this regard. However, existing YAC libraries have been made from karyotypically normal sources making the localization and cloning of specific rearrangement breakpoints much more difficult. As a solution to this problem, we present an improved method for creating YAC libraries that can utilize specialized tumor-derived materials and that can be executed effectively in a small laboratory setting. Procedures that enabled more consistent DNA insert size selection and enhanced yeast transformation frequency were employed to generate a human YAC library from a neuroepithelioma cell line containing a characteristic t(11;22) chromosomal translocation. Approximately 40,000 colonies with an average insert size of 330 kilobase pairs were created. This library was screened with two single-copy probes that bracket the translocation breakpoint. YAC clones ranging from 370 to 550 kilobase pairs that were specific for each single-copy probe were identified. Specialized YAC libraries will make many more tumor-specific chromosomal abnormalities accessible to molecular isolation.
Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.
Authors: Thibaut D , Debussche L , Blanche F
Published: May 2006

delta-Aminolevulinic acid and trimethylisobacteriochlorin are converted by cell-free protein preparations from Pseudomonas denitrificans into a metal-free pigment, precorrin-6x. This pigment, which accumulates when the cell-free system lacks NADPH, can be enzymically converted in high yield (greater than 50%) into hydrogenobyrinic acid by the complete enzyme preparation. Double-labeling experiments establish that precorrin-6x carries five C-methyl groups, which appear at C-1, C-2, C-7, C-12 alpha, and C-17 of the hydrogenobyrinic acid formed enzymically from precorrin-6x. This precursor of the corrin macrocycle is at the dehydrocorrin level of oxidation, has undergone ring contraction and extrusion of C-20, but still carries the acetic acid side chain at C-12. It is demonstrated that the conversion of precorrin-6x into hydrogenobyrinic acid specifically requires an NADPH-dependent reduction step.
Elimination of infectious human immunodeficiency virus from human T-cell cultures by synergistic action of CD4-Pseudomonas exotoxin and reverse transcriptase inhibitors.
Authors: Ashorn P , Moss B , Weinstein J N , Chaudhary V K , FitzGerald D J , Pastan I , Berger E A
Published: May 2006

We have previously described a recombinant protein, designated CD4(178)-PE40, consisting of the human immunodeficiency virus (HIV) envelope glycoprotein-binding region of human CD4 linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A. By virtue of its affinity for gp120 (the external subunit of the HIV envelope glycoprotein), the hybrid toxin selectively binds to and kills HIV-1-infected human T cells expressing surface envelope glycoprotein and also inhibits HIV-1 spread in mixed cultures of infected and uninfected cells. We now report that CD4(178)-PE40 and reverse transcriptase inhibitors exert highly synergistic effects against HIV-1 spread in cultured human primary T cells. Furthermore, combination treatment can completely eliminate infectious HIV-1 from cultures of human T-cell lines. This conclusion is based on protection of a susceptible cell population from HIV-induced killing, complete inhibition of virus protein accumulation, and elimination of HIV DNA (as judged by quantitative polymerase chain reaction analysis). The results highlight the therapeutic potential of treatment regimens involving combination of a virostatic drug that inhibits virus replication plus an agent that selectively kills HIV-infected cells.
Immunological studies of the basis for the apathogenicity of simian immunodeficiency virus from African green monkeys.
Authors: Norley S G , Kraus G , Ennen J , Bonilla J , König H , Kurth R
Published: May 2006

Potential reasons for the lack of pathogenicity of the simian immunodeficiency virus SIVagm in its natural host, the African green monkey (AGM, Cercopithecus aethiops), were investigated with respect to immunological mechanisms. The functional immune response of monkeys to infection was similar (though not identical) to that of humans to infection with human immunodeficiency virus type 1 (HIV-1). In the sera of infected animals, neutralizing antibodies were found to be low or absent, and in particular there was no neutralization of the various isolates by homologous sera. There was no detectable antibody/complement cytotoxicity, though AGM sera were able to initiate antibody-dependent cellular cytolysis of infected cells in the presence of healthy effector peripheral blood lymphocytes. As in the human/HIV system, macrophages from AGMs are readily infected by SIVagm. Two possibly important differences between the AGM/SIVagm system and the human/HIV system are (i) the low immune response of the AGMs to the core protein of SIVagm and (ii) the significantly lower inhibitory effect of SIVagm proteins on the proliferation of AGM lymphocytes.
Alpha and beta replication origins of plasmid R6K show similar distortions of the DNA helix in vivo.
Authors: Flashner Y , Shafferman A
Published: May 2006

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.
Resolution of hypervariable regions in T-cell receptor beta chains by a modified Wu-Kabat index of amino acid diversity.
Authors: Jores R , Alzari P M , Meo T
Published: May 2006

The Wu-Kabat variability coefficient is a well-established descriptor of the susceptibility of an amino acid position to evolutionary replacements. It conveniently highlights stretches of accentuated amino acid variation that, for example, in an antibody molecule account for most of the antigen contacts (complementarity-determining regions). Diverse opinion are held as to why the index yields unclear results when applied instead to the polypeptide sequences of the T-cell antigen receptor. We show that a simple modification enhances the resolving power of the index by increasing the weight on the frequency distribution of the amino acids in the formula. Application of the improved index to T-cell receptor beta chains highlights four unambiguous hypervariable regions, three of which are positioned similar to immunoglobulin complementarity-determining regions along the chain. In a Fab-like three-dimensional model of the T-cell receptor, the four hypervariable regions coincide with the four loops on the surface of the domain and form a contiguous area available for binding.
cDNA clone encoding Drosophila transcription factor TFIID.
Authors: Muhich M L , Iida C T , Horikoshi M , Roeder R G , Parker C S
Published: May 2006

Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID. A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized. The deduced amino acid sequence reveals an open reading frame of 353 residues. The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFIID and 88% identical to human TFIID. The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar. In addition, the amino-terminal region of the Drosophila TFIID contains several sequence motifs that are found in other Drosophila proteins which appear to regulate transcription.
Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription.
Authors: Kambadur R , Culotta V , Hamer D
Published: May 2006

Transcription factor IID (TFIID), the "TATA binding factor," is thought to play a key role in the regulation of eukaryotic transcriptional initiation. We have studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1. In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of wheat germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modification(s) that is required for regulated as compared to basal transcription.
Variable patterns of expression of luciferase in transgenic tobacco leaves.
Authors: Barnes W M
Published: May 2006

A carboxyl-terminally modified firefly luciferase, encoded as a gene fusion to the neomycin phosphotransferase gene (which confers kanamycin resistance), was found to be enzymatically active for both enzymes when expressed in bacteria and in transgenic plants. A military-type starlight vision system was used to conveniently analyze the pattern of gene expression in transgenic tobacco plant leaves. Transgenic tobacco plants which expressed luciferase uniformly in all areas of the leaf, and assays for luciferin, demonstrated that luciferin rapidly penetrates all regions of a tobacco leaf in at least two dimensions. Depending on the test gene structure or, presumably, on the transferred DNA (T-DNA) insertional context, other transgenic plants were obtained that expressed luciferase with a wide range of nonuniform patterns from nominally the same cauliflower mosaic virus 35S promoter. For instance, the veins can be dark, while only the interveinal regions of the leaf lamina glow, or only the small capillary veins glow, or only the major veins glow. Local and/or systemic induction in response to wounding was also demonstrated.
Modulation of function and gated learning in a network memory.
Authors: Abbott L F
Published: May 2006

Memory and learning are studied in a model neural network made from component cells with a variety of realistic intrinsic dynamic behaviors. Modulation of intrinsic cellular characteristics causes a network to switch between two entirely different modes of operation. In one mode the network acts as a selective, long-term associative memory, whereas in the other it is a nonselective, short-term latching memory. Such functional modulation can be used as a mechanism for initiating and terminating learning in a network associative memory.
Control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae.
Authors: Biswas T K
Published: May 2006

Mitochondrial promoters in Saccharomyces cerevisiae contain an identical octanucleotide [sequence: see text] sequence present just upstream of the initiation site (at the left end of the arrow). Studies have shown that the transcription rates of mitochondrial genes vary from 7- to 15-fold. The nucleotide at position +2 regulates the efficiency of mitochondrial promoters but does not affect the specificity of initiation. The data presented herein demonstrate that the variable transcription rates of mitochondrial genes are due to different levels of transcriptional initiation. The rate of first phosphodiester bond formation between a purine and a pyrimidine on a weak promoter is much lower than that of purine-purine on a strong promoter. A dinucleotide corresponding to positions +1 and +2 acts in vitro as a primer, bypassing the first phosphodiester bond formation at the initiation site. When these dinucleotides were used to prime transcription, the activities of the strong and weak promoters were found to be identical. In heparin-challenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation. These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.
Sexual hybridization of Lycopersicon esculentum and Solanum rickii by means of a sesquidiploid bridging hybrid.
Authors: DeVerna J W , Rick C M , Chetelat R T , Lanini B J , Alpert K B
Published: May 2006

A sesquidiploid hybrid having two genomes of Lycopersicon esculentum and one of Solanum lycopersicoides served as a pistillate bridging parent in crosses with Solanum rickii to produce L. esculentum x S. rickii hybrid progeny. Of the four progeny obtained, one (GH2754) was diploid and three were aneuploid with extra S. lycopersicoides chromosomes. The hybrids had morphological features of both parents, but attributes of the wild parent dominated. The hybrid nature of the four progeny was confirmed by isozyme, restriction fragment length polymorphism, and cytological analyses. A mean of 9.15 bivalents was observed in pollen mother cells of GH2754. A high level of pollen abortion was seen in all hybrids. Crosses of the hybrids with staminate S. rickii yielded one backcross individual, revealing a very low, but certain level of female fertility. Colchicine treatment of GH2754 generated one promising amphidiploid hybrid, which exhibited strong preferential chromosome pairing (94% of the examined cells had 24 bivalents) and appreciable pollen fertility (43% stainable). Chromosome pairing, isozyme, and restriction fragment length polymorphism data support a very close relationship between the two Solanum spp. and a much greater distance between them and L. esculentum, but the data do not discriminate between them in respect to their distances from the latter. The cytological and molecular observations, previous reports of successful transfer of traits from S. lycopersicoides to L. esculentum, and our hybridization of L. esculentum x S. rickii suggest good prospects for gene transfer from S. rickii to L. esculentum.
A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin.
Authors: Chaudhary V K , Gallo M G , FitzGerald D J , Pastan I
Published: May 2006

To kill human or primate cells expressing the p55 subunit of the interleukin 2 receptor, we have constructed a single-chain immunotoxin. DNA sequences encoding the first 388 amino acids of diphtheria toxin (DT) were fused to DNA elements encoding the antigen-binding portion (variable region or Fv) of the anti-Tac monoclonal antibody. The antigen-binding portion consists of 116 amino acids of the heavy-chain variable region connected by a 15-amino acid linker to 106 amino acids of the variable region of the light chain. The single-chain immunotoxin DT388-anti-Tac(Fv) was expressed in Escherichia coli and found in inclusion bodies. The monomeric form was then purified to near homogeneity with a high yield (3-5 mg/liter). Monomeric DT388-anti-Tac(Fv) was highly cytotoxic to cell lines bearing the p55 subunit of the human interleukin 2 receptor but not to cells without this subunit. DT388-anti-Tac(Fv) was also very effective in killing proliferating human T cells produced in a mixed leukocyte reaction.
Molecular clock of viral evolution, and the neutral theory.
Authors: Gojobori T , Moriyama E N , Kimura M
Published: May 2006

Evolution of viral genes is characterized by enormously high speed compared with that of nuclear genes of eukaryotic organisms. In this paper, the evolutionary rates and patterns of base substitutions are examined for retroviral oncogenes, human immunodeficiency viruses (HIV), hepatitis B viruses (HBV), and influenza A viruses. Our results show that the evolutionary process of these viral genes can readily be explained by the neutral theory of molecular evolution. In particular, the neutral theory is supported by our observation that synonymous substitutions always much predominate over nonsynonymous substitutions, even though the substitution rate varies considerably among the viruses. Furthermore, the exact correspondence between the high rates of evolutionary base substitutions and the high rates of production of mutants in RNA viruses fits very nicely to the prediction of the theory. The linear relationship between substitution numbers and time was examined to evaluate the clock-like property of viral evolution. The clock appears to be quite accurate in the influenza A viruses in man.
Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells.
Authors: Radomski M W , Palmer R M , Moncada S
Published: May 2006

Vascular endothelial cells contain a constitutive nitric oxide (NO) synthase that is Ca2(+)-dependent. In addition, we have found that these cells express, after activation with interferon-gamma and lipopolysaccharide, an inducible Ca2(+)-independent NO synthase that is distinct from the constitutive enzyme. The generation of NO by this enzyme was detectable after a lag period of 2 hr, reached a maximum between 6 and 12 hr, and was maintained for the duration of the experiment (48 hr). The expression of the inducible NO synthase was inhibited by the protein synthesis inhibitor cycloheximide, a compound that had no direct effect on the activity of either of the two enzymes. Furthermore, hydrocortisone and dexamethasone, but not progesterone, inhibited the expression of the inducible enzyme, without directly affecting the activity of either enzyme, without directly affecting the activity of either enzyme. The effect of these steroids was inhibited in a concentration-dependent manner by cortexolone, a partial agonist of glucocorticoid receptors. Thus, the inhibition of the induction of an NO synthase by glucocorticoids is a receptor-mediated event involving the inhibition of the synthesis of mRNA for de novo synthesis of this enzyme. The induction of this NO synthase may contribute to the pathophysiology of immunologically based conditions. Furthermore, the inhibition of this induction by anti-inflammatory steroids may explain some of the therapeutic and adverse effects of these compounds.
In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.
Authors: Yang N S , Burkholder J , Roberts B , Martinell B , McCabe D
Published: May 2006

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.
Analysis of interleukin 2 and various effector cell populations in adoptive immunotherapy of 9L rat gliosarcoma: allogeneic cytotoxic T lymphocytes prevent tumor take.
Authors: Kruse C A , Lillehei K O , Mitchell D H , Kleinschmidt-DeMasters B , Bellgrau D
Published: May 2006

Recombinant interleukin 2 (rIL-2) and various effector cell populations were used for adoptive immunotherapy in the Fischer strain 9L rat gliosarcoma model. The in vivo cytotoxicities of nonspecifically activated lymphocytes and specifically activated cytotoxic T lymphocytes (CTLs) were assessed in a modified in vivo neutralization (Winn) assay. Effector cells (10(6)) and 9L tumor cells (10(5] were combined with 10(4) units of rIL-2 and stereotactically implanted into the right frontal centrum semiovale of the Fischer (F344) rat. At 7 and 14 days, additional effector cells (10(6] and rIL-2 (10(4) units) were administered through the same burr hole. Nonspecifically activated splenocytes were lymphokine-activated killer (LAK) cells, both plastic-adherent and nonadherent, whereas specifically activated CTLs were either syngeneic (genetically identical) or allogeneic (genetically dissimilar). Syngeneic CTLs were T lymphocytes from Fischer rats primed in vivo with 9L cells and restimulated in vitro. Allogeneic CTLs were generated by exposing DA rat lymphocytes either to irradiated Fischer lymph node cells or to 9L Fisher tumor cells in vitro. Control groups included rats bearing 9L tumor who were untreated, those who received peripheral (i.p. or s.c.) administration of rIL-2, or those who received syngeneic unstimulated T lymphocytes and rIL-2. For a set of animals given the same inoculum of 9L tumor, significantly improved survival was shown for groups treated with nonadherent or adherent LAK cells (P less than or equal to 0.0003), syngeneic CTLs (P = 0.0327), or allogeneic CTLs (P = 0.0025) over untreated control animals by using Mantel-Haenzel nonparametric logrank equations. Only treatment with allogeneic CTLs prevented tumor take.
Excision in algebraic K-theory and Karoubi's conjecture.
Authors: Suslin A A , Wodzicki M
Published: May 2006

We prove that the property of excision in algebraic K-theory is for a Q-algebra A equivalent to the H-unitality of the latter. Our excision theorem, in particular, implies Karoubi's conjecture on the equality of algebraic and topological K-theory groups of stable C*-algebras. It also allows us to identify the algebraic K-theory of the symbol map in the theory of pseudodifferential operators.