bluuebunny/crossref_metadata_2025 huggingface dataset:
Rod light response augmented by active phosphodiesterase.
Authors: Shimoda Y , Hurley J B , Miller W H
Published: May 2006

Light activates rod outer segment (ROS) phosphodiesterase (PDEase), as shown by previous biochemical and physiological studies. We have further investigated the role of PDEase in this system by injecting trypsin-activated PDEase, purified from bovine ROS, into single ROS of the isolated retina of the toad Bufo marinus. Injection of about 300 molecules of activated PDEase in darkness is without immediate detectable effect, as measured by intracellular membrane-voltage recording. The effect of the activated PDEase injections only becomes evident after illumination. The light response is augmented; kinetics of repolarization are slowed. We conclude that this augmentation of the light-dependent hyperpolarization results from the hydrolysis of endogenous cyclic GMP caused by injected PDEase. These results provide evidence that PDEase affects light-dependent channels of the vertebrate scotopic photoreceptors but do not specify whether the effects are exercised for the initial hyperpolarizing phase of the receptor potential and for the recovery phase or only for the recovery phase.
The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.
Authors: Hartzell S W , Byrne B J , Subramanian K N
Published: May 2006

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.
Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.
Authors: Tsai P K , Ballou L , Esmon B , Schekman R , Ballou C E
Published: May 2006

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core.
Genomic constitution of an H-2:Tla variant leukemia.
Authors: Shen F W , Chaganti R S , Doucette L A , Litman G W , Steinmetz M , Hood L , Boyse E A
Published: May 2006

A TL+ leukemia of a (B6 X A)F1 hybrid mouse (H-2b/H-2a) was previously subjected to immunoselection against H-2a by passage in (B6 X A.SW)F1 mice (H-2b/H-2s). A variant leukemia line was obtained that serologically lacked not only the H-2a phenotype but also the TL phenotype determined by the linked cis Tlaa allele of strain A. The H-2b phenotype and the TL phenotype of the Tlab allele of the B6 strain, which is expressed only by leukemia cells, were retained by the variant. Southern blotting with an H-2 cDNA probe that identifies restriction fragment polymorphisms distinguishing alleles of the H-2 and Tla regions of the B6 and A strains indicates that both the H-2a and Tlaa alleles are missing from the genome of this H-2a:Tlaa negative variant. Since the variant has two apparently unaltered chromosomes 17, where the H-2:Tla complex is situated, and since the intensity of bands in Southern blotting is suggestive of H-2b homozygosity, it is considered that loss of the H-2a:Tlaa haplotype by the variant was accompanied by duplication of the H-2b:Tlab haplotype. The implied change from heterozygosity to homozygosity that the variant has undergone with respect to H-2:Tla was not paralleled by a similar change at the three other loci tested, since the variant retained heterozygosity for Pep-3 (chromosome 1), Gpi-1 (chromosome 7), and Es-1 (chromosome 8).
Calcium-binding proteins in human carcinoma cell lines.
Authors: Pfyffer G E , Haemmerli G , Heizmann C W
Published: May 2006

Elevated levels of Ca2+-binding proteins are reported in transformed cells and thought to be involved in their uncontrolled proliferation and often increased motility. Therefore, three cell lines [LICR(Lond)-HN 1, -HN 2, and -HN 6] derived from human carcinomas displaying various degrees of locomotive activity were investigated for the presence of parvalbumin and related Ca2+-binding proteins. By applying different immunohistochemical methods in conjunction with a monospecific anti-parvalbumin antiserum, an intense staining was seen in cells displaying translocative motility. Often in these cells, an association with filamentous structures located in the nuclear region was observed. Unique Ca2+-binding proteins, absent from comparable normal tissue, were found in the malignant cell lines when analyzed by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatography. From these tumor cells a protein (Mr, 12,000; pI, 4.8) was isolated that crossreacted with antiparvalbumin antiserum. Peptide maps of this protein revealed a further structural homology to parvalbumin (Mr, 12,000; pI, 4.9). In analogy to muscle, where there is evidence for a regulatory role of parvalbumin in the contraction-relaxation cycle, we speculate that this protein is tumor-associated and connected to the motile behavior of carcinoma cells.
Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein.
Authors: Paterson R G , Harris T J , Lamb R A
Published: May 2006

The nucleotide sequence of the mRNA coding for the fusion glycoprotein (F) of the paramyxovirus, simian virus 5, has been obtained. There is a single large open reading frame on the mRNA that encodes a protein of 529 amino acids with a molecular weight of 56,531. The proteolytic cleavage/activation site of F, to yield F2 and F1, contains five arginine residues. Six potential glycosylation sites were identified in the protein, two on F2 and four on F1. The deduced amino acid sequence indicates that F is extensively hydrophobic over the length of the polypeptide chain. Three regions are very hydrophobic and could interact directly with membranes: these are the NH2-terminal putative signal peptide, the COOH-terminal putative membrane anchorage domain, and the NH2-terminal region of F1.
Sequences homologous to P elements occur in Drosophila paulistorum.
Authors: Daniels S B , Strausbaugh L D , Ehrman L , Armstrong R
Published: May 2006

P elements, a class of mobile genetic elements that cause hybrid dysgenesis in Drosophila melanogaster, have thus far been shown to occur only in this species. Using whole genome blot analysis, we present evidence which indicates that sequences homologous to D. melanogaster P elements also occur in Drosophila paulistorum, a distant relative. D. paulistorum is considered a species complex, consisting of six known incipient species or semispecies. All six semispecies possess P element homologous sequences. We further show that there is conservation of restriction enzyme recognition sites between the D. melanogaster and D. paulistorum P element sequences. The presence of P elements in these two species may clarify the roles of recent invasion and rapid loss in the temporal distribution of P elements in Drosophila.
Understanding biological computation: reliable learning and recognition.
Authors: Hogg T , Huberman B A
Published: May 2006

We experimentally examine the consequences of the hypothesis that the brain operates reliably, even though individual components may intermittently fail, by computing with dynamical attractors. Specifically, such a mechanism exploits dynamic collective behavior of a system with attractive fixed points in its phase space. In contrast to the usual methods of reliable computation involving a large number of redundant elements, this technique of self-repair only requires collective computation with a few units, and it is amenable to quantitative investigation. Experiments on parallel computing arrays show that this mechanism leads naturally to rapid self-repair, adaptation to the environment, recognition and discrimination of fuzzy inputs, and conditional learning, properties that are commonly associated with biological computation.
A product of yeast RAS2 gene is a guanine nucleotide binding protein.
Authors: Tamanoi F , Walsh M , Kataoka T , Wigler M
Published: May 2006

Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.
Regulation of ribosomal RNA promoters with a synthetic lac operator.
Authors: Brosius J , Holy A
Published: May 2006

A synthetic 21-base-pair long DNA fragment containing the central lac operator sequence has been inserted near the initiation point of the cloned Escherichia coli rrnB rRNA promoter P2 in the natural and reverse orientation. RNA synthesis is efficiently repressed in both orientations in lac Iq strains and is induced with isopropyl beta-D-thiogalactoside. When the rrnB promoter P1 is also present, upstream from P2 and the synthetic lac operator, repression of transcription is incomplete. The levels of transcription were measured in vivo, indirectly by the expression of a protein (chloramphenicol acetyltransferase), or directly by the expression of a stable RNA (E. coli 4.5S RNA) in a simple assay involving gel electrophoresis of unlabeled total RNA from E. coli. The rrnB promoter constructions can produce high levels of protein expression as well as high levels of expression of stable RNA.
A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia.
Authors: Pegoraro L , Palumbo A , Erikson J , Falda M , Giovanazzo B , Emanuel B S , Rovera G , Nowell P C , Croce C M
Published: May 2006

We have established a cell line, which we named 380, from a young male with acute lymphoblastic leukemia (FAB type L2). Karyologic analysis of this cell line indicates that it carries an 8;14 and a 14;18 chromosome translocation, which are characteristic of Burkitt lymphoma and of follicular lymphoma, respectively. This cell line is Epstein-Barr virus antigen-negative, reacts with monoclonal antibodies specific for B cells, and contains rearranged immunoglobulin heavy and light chain genes, but does not express human immunoglobulins. In this cell line, both mu heavy chain constant (C mu) loci are rearranged within the joining (JH) DNA segment. One of the JH segments on one of the 14q+ chromosomes is rearranged with a segment of chromosome 8, where the c-myc oncogene resides, while the other is rearranged with a segment of chromosome 18 where a putative oncogene, which we have called bcl-2, is located. The c-myc oncogene, which is translocated to one of the 14q+ chromosomes, is in its germ-line configuration more than 14 kilobases away from both the JH segment and the heavy chain enhancer that is located between the JH and mu switch region. Based on these findings, we propose a model of some aspects of B-cell oncogenesis according to which B-cell neoplasms carrying translocations involving the heavy chain loci on both human chromosomes 14 are the result of a multiple step process.
Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.
Authors: Sottrup-Jensen L , Folkersen J , Kristensen T , Tack B F
Published: May 2006

Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier.
Requirement for protein synthesis in the regulation of a circadian rhythm by serotonin.
Authors: Eskin A , Yeung S J , Klass M R
Published: May 2006

Serotonin (5-HT, 5-hydroxytryptamine) regulates the phase of a circadian pacemaker located within the eye of Aplysia. We are attempting to define the cellular and biochemical events involved in the regulatory pathway through which serotonin acts. Previously, we have shown that an activation of adenylate cyclase and an increase in cAMP are events in the 5-HT phase-shifting pathway. In this paper, we examine the role of protein synthesis in mediating the effect of 5-HT and cAMP on the phase of the circadian rhythm. Exposure of eyes to anisomycin, an inhibitor of protein synthesis, completely blocked the advance shift in phase produced by 5-HT. Although anisomycin by itself can produce phase shifts, it did not affect the rhythm at the phases where the blocking experiments were performed. The specificity of action of anisomycin was investigated in two ways. First, deacetylanisomycin, an analogue of anisomycin that is inactive in inhibiting protein synthesis, did not affect the shift in phase produced by 5-HT. Second, anisomycin did not inhibit two other effects of 5-HT on the eye that also appear to be mediated by cAMP: an inhibition of spontaneous optic nerve activity and an increase in the photosensitivity of the eye. The step in the 5-HT phase-shifting pathway that is sensitive to anisomycin appears to occur after the cAMP step because anisomycin also inhibits the ability of 8-benzylthio-cAMP to shift the phase of the rhythm. We have also examined whether 5-HT directly regulates the synthesis of any proteins in the eye. Using two-dimensional gel electrophoresis, we have found that 5-HT appears to increase the synthesis of a protein with an apparent molecular weight of 67,000. Our results indicate that protein synthesis is necessary for 5-HT to shift the phase of the rhythm and that 5-HT appears to regulate the expression of at least one protein in the eye.
Identification of the alpha beta monomer of the adipocyte insulin receptor by insulin binding and autophosphorylation.
Authors: Velicelebi G , Aiyer R A
Published: May 2006

The insulin receptor consists of an insulin-binding subunit (alpha) of 135,000 daltons. More recently, it has been documented that the receptor undergoes insulin-stimulated autophosphorylation that predominantly labels a 95,000-dalton (beta) subunit. We solubilized rat adipocyte insulin receptors in Triton X-100 and partially purified the protein on a wheat germ agglutinin-Sepharose affinity column. Subsequently, we labeled the two subunits of the receptor independently by using 125I-labeled insulin for the 135,000-dalton alpha-subunit and 32P for the 95,000-dalton beta-subunit. Sucrose density gradient sedimentation and NaDodSO4/PAGE were used to characterize the native, oligomeric structure of the receptor. In 0.1% Triton X-100, the receptor sedimented as a single peak of s20,w = 10.2 S as detected by both 125I and 32P. NaDodSO4/PAGE under nonreducing conditions revealed a large species that appeared to be alpha 2 beta 2 and, to a lesser extent, alpha beta. Treatment of the solubilized, partially purified receptor with 10 mM dithiothreitol led to the partial conversion of the 10.2S species to a smaller one sedimenting at 6.6 S. The composition of this species was determined to be alpha beta by nonreducing NaDodSO4/PAGE. Our results suggest that detergent-solubilized insulin receptors can exist as dimers and monomers. The oligomeric structure of receptors functional in the cell membrane cannot be immediately deduced from these results due to the possibility of artifacts arising from membrane disruption and extraction procedures. However, the ability to label the two subunits of the receptor separately should facilitate a detailed study of its oligomeric structure both in solution and in the membrane.
Coordinate replication of members of the multigene family of core and H1 human histone genes.
Authors: Iqbal M A , Plumb M , Stein J , Stein G , Schildkraut C L
Published: May 2006

Cells of the K562 human erythroleukemia cell line were obtained in different stages of the cell cycle by centrifugal elutriation. The cells had been previously labeled for 2 hr with BrdUrd so that BrdUrd-DNA synthesized during four different selected intervals of the S phase could be isolated. This DNA was used to determine the temporal replication during S phase of EcoRI segments containing histone genes. Cloned human genomic segments containing the core histone genes (H2A, H2B, H3, and H4), H2A and H2B pseudogenes, and the H1 gene were prepared. The genomic inserts were excised from these plasmids, nick-translated, and used as hybridization probes. The results with different probes compared on the same and on independently prepared DBM-paper transfers indicate that all of these histone genes replicate during the first half of the S phase. These genes were not among the earliest to replicate in the K562 cell line. Similar studies were carried out with HeLa cells in which EcoRI segments containing the H4 histone and H2A and H2B pseudogenes were found to replicate during the first half of the S phase. These histone genes replicate during the interval of the S phase when histone mRNA appears in the cytoplasm at the maximal rate. The possible relationship between these events is discussed.
Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.
Authors: O'Connor C M , Aswad D W , Clarke S
Published: May 2006

Two purified isozymes of protein carboxyl methyltransferase from bovine brain catalyze the substoichiometric transfer of methyl groups in vitro from S-adenosyl-L-[methyl-3H]methionine to several erythrocyte membrane proteins, which include bands 2.1, 3, and 4.1, as well as several integral membrane polypeptides. D-Aspartic acid beta-[3H]methyl ester has been isolated from proteolytic digests of these methylated proteins, suggesting that protein D-aspartyl residues can serve as methyl-acceptor sites for the two brain enzymes. This formation of D-aspartic acid beta-[3H]methyl ester is competitively inhibited by the peptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, which contains an L-aspartyl residue in an unusual beta-peptide linkage. Since this peptide is a stoichiometric substrate for the brain methyltransferases, it appears that one enzymatic activity can catalyze methyl ester formation at both D-aspartyl and L-isoaspartyl sites. In these respects, the activity of both brain isozymes closely resembles those previously described for the erythrocyte enzyme. The results are discussed in terms of a model in which derivatized aspartyl residues in proteins, arising by either racemization or isomerization, are recognized by the methyltransferase; the enzyme may function in either the metabolism or correction of the altered structures. The presence of a similar enzyme in both translationally active (brain) and inactive (erythrocyte) tissues suggests that the reactions are of general importance to cellular integrity.
Evidence for an intracellular precursor for human B-cell growth factor.
Authors: Sahasrabuddhe C G , Morgan J , Sharma S , Mehta S , Martin B , Wright D , Maizel A
Published: May 2006

Human B-cell growth factor has been described as a trypsin-sensitive protein of Mr 12,000-14,000. Evidence is provided herein that this relatively low molecular weight product may be released from a larger precursor molecule of Mr 60,000-80,000. The precursor protein is confined to the cytosol of freshly isolated T lymphocytes, and only the Mr 12,000-14,000 moiety is released upon lectin stimulation. The precursor protein was subjected to limited tryptic digestion, which demonstrated that the biologically active fraction of the moiety resided in a relatively low molecular weight fragment. The T lymphocyte routinely possessed an intracytoplasmic pool of the precursor protein, the amount of which cyclically varied depending upon its depletion by the secretion process of a lower molecular weight product. Analysis of the mRNA size coding for the majority of B-cell growth factor activity, determined by translation in Xenopus laevis oocytes, suggested that the B-cell growth factor-specific mRNA resided in the greater than or equal to 15S range. This value is consistent with the size of the larger precursor. Therefore, it is proposed that a precursor-product relationship exists for the processing of human B-cell growth factor, analogous to that which has been described for several other cytokines.
Human antibodies to a Mr 155,000 Plasmodium falciparum antigen efficiently inhibit merozoite invasion.
Authors: Wåhlin B , Wahlgren M , Perlmann H , Berzins K , Björkman A , Patarroyo M E , Perlmann P
Published: May 2006

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, less than 1 microgram/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.
Physiological role of alpha-melanocyte-stimulating hormone in modulating the secretion of prolactin and luteinizing hormone in the female rat.
Authors: Khorram O , Bedran de Castro J C , McCann S M
Published: May 2006

Long-term ovariectomized (OVX) rats were injected in the third cerebral ventricle with 5 microliter of the globulin fraction of an antiserum raised against alpha-melanocyte-stimulating hormone (alpha-MSH) or an equal volume of the globulin fraction of normal rabbit serum (NRS). Immunoneutralization of brain alpha-MSH produced an increase in the area under the secretion curve of prolactin (Prl), the amplitude of Prl pulses, and mean plasma Prl (P less than 0.01). In animals that had received two injections of NRS or anti-MSH and were subjected to a 2-min ether stress, Prl levels significantly increased within 5 minutes in the NRS-injected rats, whereas Prl levels in the antiserum-injected rats did not increase any further from the initially high baseline levels. The administration of antibodies against alpha-MSH produced a small increase (P less than 0.05) in the area under the secretion of luteinizing hormone (LH) and mean plasma LH; however, the number of LH pulses was unaffected. We conclude that endogenous alpha-MSH of central origin is a physiological neuromodulator of release of Prl and LH in the OVX rat and is involved in the stress-induced release of Prl.
On the construction of nonequatorial minimal hyperspheres in

S
n

(1) with stable cones in R

n
+1
Authors: Hsiang Wu-yi , Sterling Ivan
Published: May 2006

Within the framework of equivariant differential geometry, we outline the construction of some imbedded minimal hyperspheres of

S
n

(1) and show that many of them have a stable cone in R

n
+1

. The statement of these and related results are given.
Intracellular lymphocyte activation and carrier-mediated transport of C8-substituted guanine ribonucleosides.
Authors: Goodman M G , Weigle W O
Published: May 2006

The studies described in this report constitute evidence substantiating that certain exogenous nucleoside derivatives can activate lymphocytes by acting intracellularly. These molecules, the C8-substituted guanine ribonucleosides, have recently been demonstrated to exert potent immunostimulatory and immunoregulatory activities both in vitro and in vivo. The current studies were undertaken to investigate whether the site of induction of mitogenesis in murine B lymphocytes by these compounds was intracellular or at the plasma membrane. Uptake of 8-bromoguanosine was found to proceed by carrier-mediated transport. Like that described for adenosine, the uptake system for 8-bromoguanosine could be resolved into high-affinity and low-affinity components. The hypothesis that the C8-substituted guanine ribonucleosides act intracellularly was tested in several ways. Immobilization of these substituted nucleosides, either on Sepharose beads or in the form of high molecular weight polymers, resulted in total loss of their mitogenicity. In addition, maneuvers designed to diminish plasma membrane fluidity interfered with transmembrane signaling by surface membrane-directed mitogens far more than they did with activation by the substituted nucleosides. Furthermore, modulation of surface membrane protein (IgM) with anti-IgM antibodies similarly resulted in differential inhibition of transmembrane signals with relatively little effect on activation by 8-mercaptoguanosine. Taken together, these data are consistent with the hypothesis that the C8-substituted guanine ribonucleosides trigger the cell at an intracellular site.
Serotonin binds specifically and saturably to an actin-like protein isolated from rat brain synaptosomes.
Authors: Small D H , Wurtman R J
Published: May 2006

A soluble serotonin-binding protein was identified in a high-speed supernatant fraction of an osmotically shocked rat brain synaptosome (P2) preparation. The binding of serotonin was saturable (Bmax = 6.0 nmol per mg of protein) and was specific for serotonin and a few structurally related compounds including dopamine and norepinephrine. Binding of serotonin (1 microM) was inhibited approximately equal to 40% by chlorpromazine (10 microM). The affinity of serotonin for the binding protein was low in the crude extract (Kd = 1.7 X 10(-3)M). However, on purification by chromatography on a column of phenothiazine agarose, a higher affinity (Kd = 10(-5) M) binding component was also observed. The purified protein was greatly enriched in a polypeptide of Mr of 43,000 that comigrated on polyacrylamide gel with skeletal muscle actin. Muscle actin also bound serotonin, and the binding to actin was similar to that of the purified protein in both the specificity of the binding and the affinity for serotonin. It is likely that the serotonin-binding protein is identical to cytoplasmic G-actin or an actin-like protein of similar molecular weight.
Molecular analysis of the Neurospora qa-1 regulatory region indicates that two interacting genes control qa gene expression.
Authors: Huiet L
Published: May 2006

The qa-1 regulatory region controls the expression of the three structural genes required for the early reactions in quinic acid catabolism in Neurospora crassa. Genetic analysis previously identified two types of noninducible qa-1 mutants, qa-1S and qa-1F, which mapped in separate non-overlapping regions. These mutations were originally interpreted as defining separate domains of a single regulatory protein. This communication describes the further genetic and physical characterization of the qa-1 regulatory region. Using both Neurospora transformation and DNA . RNA hybridization, it has been shown that the qa-1 region consists of two distinct genes corresponding to the two original mutational types qa-1S and qa-1F. The analysis of the mRNA species hybridizing to these regions indicates that the qa-1F gene encodes a 2.9-kilobase (kb) mRNA, while the qa-1S gene encodes related 4.1-kb and 3.4-kb mRNAs. The transcriptional regulation of one of these genes, qa-1S, was examined. Evidence is presented that the qa-1S gene is induced by quinic acid and is also subject to apparent autogenous regulation as well as to control by the qa-1F gene product. Based on these results and earlier genetic analysis, the hypothesis is proposed that one of the two qa regulatory genes encodes a repressor protein (qa-1S), and the other encodes an activator protein (qa-1F), both of which control qa gene expression.
Distinct mechanisms may account for the growth-promoting activity of interleukin 3 on cells of lymphoid and myeloid origin.
Authors: Palacios R , Garland J
Published: May 2006

We have investigated whether interleukin 3 (IL-3) supports the growth of cells of different lineages by the same mechanism(s). The experiments were carried out with Ea3 cells, a mouse pre-B cell line, and S-480-3 cells, a mouse basophil cell line, both of which are totally IL-3 dependent. We found that Ea3 lymphocytes but not S-480-3 basophils absorb partially purified IL-3. Both Ea3 and S-480-3 cells respond to IL-3 by increasing anaerobic glycolysis as determined by lactic acid production. S-480-3 cells responded to exogenous ATP by maintaining proliferation and reducing lactic acid production, but Ea3 lymphocytes are refractory to exogenous ATP. We conclude that there may be two distinct mechanisms by which cells respond to IL-3, indicated by early events concerning the binding of IL-3 and the effect of exogenous ATP on respiratory metabolism. One appears to be a ligand-receptor-mediated mechanism in lymphoid cells and the other to be a mechanism that is partially replaceable by exogenous ATP in nonlymphoid cells not associated with lymphoid-like receptors. Our findings may explain (i) the apparent variety of cell lineages promoted by IL-3 by a widely available mechanism that supports glycolysis and, therefore, enables both proliferation and possibly expression of binding sites for lineage specific differentiation factors and (ii) the existence of lymphocytes that express receptors specific for IL-3 and are inducible for other characteristics and functions in a regulated manner.
Plasma membrane biogenesis in eukaryotic cells: translocation of newly synthesized lipid.
Authors: Mills J T , Furlong S T , Dawidowicz E A
Published: May 2006

We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag, in a linear manner up to 1 hr. Labeled sterol and phospholipids appeared in the plasma membrane, after a 30-min lag, at approximately the same rate. However, the ratio of newly synthesized sterol to phospholipid was significantly enriched in the plasma membrane relative to the whole cell, even at the earlier time points. Pulse-chase experiments indicated that sterols and phospholipids are turned over in the plasma membrane with similar, rather short half-lives. The results of these studies suggest that, although sterols and phospholipids are transported to the cell surface with similar kinetics, some sorting of the lipids must occur at an early stage in membrane biogenesis. The data are consistent with a model of lipid translocation by vesicular transport.
Selective killing of Fc-receptor-bearing tumor cells through endocytosis of a drug-carrying immune complex.
Authors: Shen W C , Ryser H J
Published: May 2006

A soluble immune complex was used as a drug carrier targeted to Fc-receptor-positive cells. Two receptor-positive tumor cell lines, WEHI-3 and M5076, were exposed to methotrexate-human serum albumin conjugate (MTX-HSA) in the presence and absence of anti-HSA antiserum. Both cell types were killed by 30 nM MTX when the drug conjugate was given in the presence of antiserum but were totally unaffected in the absence of antiserum. Drug-free HSA given with antiserum had no effect. Both cell lines responded similarly despite their marked difference in phagocytotic activity. One of the two lines, M5076, is defective in MTX transport and hence resistant to free MTX. Since this line would not be affected by MTX released extracellularly from MTX-HSA, its susceptibility implies that MTX is released inside cells, after endocytosis of the complex, and that endocytosis circumvents the transport defect. Two cell lines lacking Fc receptors (CHO and L929) were not influenced by the drug complex. The pharmacologic effect is mediated by a specific ligand-receptor interaction, since Fc receptor-positive cells are protected by an excess of unconjugated HSA and by the addition of a small amount of staphylococcal protein A, which binds to the Fc portion of IgG. These data demonstrate that Fc receptors can be exploited for cellular drug delivery using a common antigen-antibody complex as a drug carrier.
Tubular arrays of the actin-DNase I complex induced by gadolinium.
Authors: Fowler W E , Buhle E L , Aebi U
Published: May 2006

We describe the preparation and structural analysis of ordered tubular arrays of the actin-DNase I complex. These structures consist of helically stacked rings; each ring is 73 A thick, has a 240 A outer and a 120 A inner diameter, and has 7-fold rotational symmetry. The actin-DNase I complex forms tubes under conditions in which actin alone aggregates into crystalline sheets-i.e., in the presence of the trivalent cation gadolinium. Moreover, upon addition of an equimolar amount of DNase I, crystalline actin sheets are slowly converted to tubes. The rings making the tubes contain a radial dyad axis that may be identical to the dyad axis of the unit cell of the crystalline actin sheet. Evidence is presented for this identification, which in turn allows tentative assignment of actin- and DNase I-containing regions in three-dimensional reconstructions of the rings. The structural analysis presented here may be useful in aligning available three-dimensional molecular models of actin determined from crystals of the actin-DNase I complex and from crystalline actin sheets with each other and ultimately within the biologically important actin filament.
Nucleotide sequence of the tms genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis.
Authors: Klee H , Montoya A , Horodyski F , Lichtenstein C , Garfinkel D , Fuller S , Flores C , Peschon J , Nester E , Gordon M
Published: May 2006

The nucleotide sequence of the tumor morphology locus, tms, from pTiA6NC has been determined. The sequence analysis indicates that each of two polyadenylylated transcripts encoded by this locus contains an open reading frame; the predicted transcript 1 gene product has a molecular size of 83,769 daltons, and the predicted transcript 2 gene product, of 49,588 daltons. The precise start and stop positions of the transcript 2 RNA have been mapped with S1 nuclease. Several insertion mutations have been constructed. One of these localizes the transcript 2 promoter within the 72 base pairs 5' to transcription initiation. Significant homology was observed between the protein encoded by transcript 1 and the adenine binding region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, suggesting that the transcript 1 protein binds adenine either as substrate or cofactor.
Yields of Soviet underground nuclear explosions from seismic surface waves: Compliance with the Threshold Test Ban Treaty
Authors: Sykes Lynn R. , Cifuentes Inés L.
Published: May 2006

Magnitudes of the larger Soviet underground nuclear weapons tests from the start of the Threshold Test Ban Treaty in 1976 through 1982 are determined for short- and long-period seismic waves. Yields are calculated from the surface wave magnitude for those explosions at the eastern Kazakh test site that triggered a small-to-negligible component of tectonic stress and are used to calibrate body wave magnitude-yield relationship that can be used to determine the sizes of other explosions at that test site. The results confirm that a large bias, related to differential attenuation of P waves, exists between Nevada and Central Asia. The yields of the seven largest Soviet explosions are nearly identical and are close to 150 kilotons, the limit set by the Threshold Treaty.
Proviruses are adjacent to c-myc in some murine leukemia virus-induced lymphomas.
Authors: Steffen D
Published: May 2006

Sixty-one murine leukemia virus-induced lymphomas were examined for evidence of proviral insertions adjacent to known oncogenes. Five of these lymphomas were found to have alterations adjacent to c-myc. Two of the lymphomas with altered c-myc sequences were examined in detail. Evidence was obtained showing that the alterations in the c-myc sequences in these two lymphomas were the result of proviral integrations. This result suggests that lymphomagenesis by murine leukemia viruses can result from insertional mutagenesis of cellular oncogenes.
Identification of novel neural- and neural retina-specific antigens with a monoclonal antibody.
Authors: Cole G J , Glaser L
Published: May 2006

A fluorescence-activated cell sorter screening method has been used to identify hybridomas that secrete monoclonal antibodies that can bind to viable subpopulations of embryonic chicken neural retina cells. One monoclonal antibody, C1H3, recognizes two nervous tissue-specific polypeptides that exhibit distinct developmental patterns. The monoclonal antibody reacts with a 140-kilodalton (kDa) polypeptide that is present at early stages of development (day 7) but is detected by immunoblotting in only negligible amounts at later times (day 17). In contrast, a 170-kDa polypeptide is first detectable by immunoblotting at day 10 and is the predominant C1H3 antigen at day 17. Analysis of proteolytic fragments of the two proteins indicates that the polypeptides are distinct molecules that share a common antigenetic determinant. Both polypeptides are neural-specific; the 140-kDa polypeptide appears to be retina-specific, while the 170-kDa polypeptide is also present in other areas of the nervous system. Metabolic labeling of retina cells in situ at early embryonic stages reveals only the synthesis of the 140-kDa polypeptide. When such cells are dissociated and labeled in vitro, they synthesize primarily the 170-kDa polypeptide. Thus, the differential rate of synthesis of these two polypeptides is controlled by environmental factors that possibly include cell-cell contacts or an unknown systemic factor. The 140-kDa polypeptide is a unique marker for early neural retina cells.
Retinal migration during dark reduction of bacteriorhodopsin
Authors: Wolber Paul K. , Stoeckenius Walther
Published: May 2006

When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, ≈45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys-216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the ε-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine ε-amino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin.
Hematopoietic cell transformation by a murine recombinant retrovirus containing the src gene of Rous sarcoma virus.
Authors: Pierce J H , Aaronson S A , Anderson S M
Published: May 2006

A recombinant murine retrovirus (MRSV) containing the src gene of avian Rous sarcoma virus (RSV) was shown to induce hematopoietic colonies in infected mouse bone marrow. MRSV-induced colony formation followed single-hit kinetics and required mercaptoethanol in the agar medium. Cells from the colonies induced by MRSV could be established as continuous cell lines that demonstrated unrestricted self-renewal in vitro and tumorigenicity in vivo. The transformants, all of which expressed high levels of the Rous sarcoma virus transforming protein, pp60src, appeared to be at an early stage in lymphoid cell differentiation. They lacked Fc receptors and detectable immunoglobulin mu heavy chain synthesis, markers normally associated with committed B cells. The majority of the MRSV-transformed cell lines contained high levels of terminal deoxynucleotidyl transferase, an enzyme present in lymphoid progenitor cells committed to the T-cell lineage. One cell line expressed Thy-1 antigen, but none expressed Lyt-1 and Lyt-2, markers of more differentiated T cells. These findings demonstrate that the src gene is capable of transforming cells of hematopoietic origin.
Characterization of a herpes simplex virus type 1 mutant resistant to benzhydrazone, a selective inhibitor of herpesvirus glycosylation.
Authors: Tognon M , Manservigi R , Cavrini V , Campadelli-Fiume G
Published: May 2006

Benzhydrazone [BH; 1H-benz[f]indene-1,3(2H) -dionebis(amidinohydrazone)] significantly inhibits glycosylation of proteins, but only in cells infected with herpes simplex virus. We report on a herpes simplex virus type 1 (HSV-1) mutant resistant to BH. A syncytium-inducing mutant designated HSV-1(13)S11 was found to be biochemically resistant to BH in that [14C]glucosamine incorporation was not inhibited in infected HEp-2 cells exposed to the drug. Intertypic recombinants were obtained which showed that BH resistance is encoded in the DNA of the mutant virus and may be transferred into the genome of BH-sensitive HSV. In the recombinants the biochemical resistance marker segregated from the syncytial marker, suggesting that the two markers probably map in different loci. The BH-resistant mutant did not complement wild-type BH-sensitive HSV-1 and -2. Furthermore, resistance was apparent in HEp-2 but not in Vero cells. The paper discusses the hypothesis that inhibition of glycosylation of HSV proteins is the consequence of modification or selective transport of BH involving a HSV gene product.
Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera.
Authors: Stahl H D , Coppel R L , Brown G V , Saint R , Lingelbach K , Cowman A F , Anders R F , Kemp D J
Published: May 2006

We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera. This procedure can be used for two distinct purposes. First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures. This requires that the antigen can be expressed in E. coli and that antisera are available that differ substantially in their reactivities to the component of interest. To develop the procedure, we used two polyspecific sera that shared many anti-P. falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P. falciparum strain FCQ27/PNG (called FC27). Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences. Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone. The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.