bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of interferon action: structure of the mouse PKR gene encoding the interferon-inducible RNA-dependent protein kinase.
Authors: Tanaka H , Samuel C E
Published: May 2006

The gene for the RNA-dependent eIF-2 alpha protein kinase (PKR) was isolated from mouse genomic DNA and characterized. The mouse PKR gene contains 16 exons and spans about 28 kilobase pairs. Exon 1 is untranslated; the AUG translation initiation site is located early in the second exon. Exon 16 includes the UAG translation termination site. ATTAAA polyadenylylation signal, and a putative TA rather than CA 3' cleavage site. Primer extension analysis determined one major as well as multiple minor transcription initiation sites; the major site was 159 bp upstream of the translation initiation site. The complete cDNA of mouse PKR is, therefore, 2334 bp in length excluding the 3' poly(A)+ tail. The PKR gene 5' flanking region was a functional promoter in interferon-treated, transfected cells as measured with chloramphenicol acetyltransferase as the reporter gene. Sequence analysis of the 5' flanking region disclosed numerous potential binding sites for transcription factors including both an ISRE element and a GAS element involved in interferon inducibility; Ets, Myb, MyoD, and E2F sites commonly associated with growth control regulation and differentiation; and NF-kappa B-like sites as well as sites for two types of interleukin 6-activated factors, NF-IL6 and APRF, often associated with acute-phase, immune, and inflammatory response genes.
Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.
Authors: Kunz B A , Henson E S , Roche H , Ramotar D , Nunoshiba T , Demple B
Published: May 2006

The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apn1, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G.C pair was increased 10-fold in Apn1-deficient yeast, including a 59-fold increase in the rate of A.T-->C.G events. In contrast, the rate of transversion to an A.T pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cerevisiae. Other results presented here show that AP endonuclease-deficient Escherichia coli exhibit a mutator phenotype consistent with the A-rule.
Structure of homeobox-leucine zipper genes suggests a model for the evolution of gene families.
Authors: Schena M , Davis R W
Published: May 2006

Homeobox genes are present in both plants and animals. Homeobox-leucine zipper genes, however, have been identified thus far only in the small mustard plant Arabidopsis thaliana. This observation suggests that homeobox-leucine zipper genes evolved after the divergence of plants and animals, perhaps to mediate specific regulatory events. To better understand this gene family, we isolated several sequences containing the homeobox-leucine zipper motif and carried out a comparative analysis of nine homeobox-leucine zipper genes (HAT1, HAT2, HAT3, HAT4, HAT5, HAT7, HAT9, HAT14, and HAT22). Gene structures, sequence comparisons, and chromosomal locations suggest a simple model for the evolution of these genes. The model postulates that a primordial homeobox gene acquired a leucine zipper by exon capture. The nascent homeobox-leucine zipper gene then appears to have undergone a series of gene duplication and chromosomal translocation events, leading to the formation of the HAT gene family. This work has general implications for the evolution of regulatory genes.
Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity.
Authors: Atkinson E M , Palcic M M , Hindsgaul O , Long S R
Published: May 2006

Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.
Striate cortex extracts higher-order spatial correlations from visual textures.
Authors: Purpura K P , Victor J D , Katz E
Published: May 2006

Spatial correlations define the statistical structure of any visual image. Two-point correlations inform the visual system about the spatial frequency content of an image. Higher-order correlations can capture salient features such as object contours. We studied "isodipole" texture discrimination in V1 to determine if higher-order spatial correlations can be extracted by early stages of cortical processing. We made epicortical, local field potential, and single-cell recordings of responses elicited by isodipole texture interchange in anesthetized monkeys. Our studies demonstrate that single neurons in V1 can signal the presence of higher-order spatial correlations in visual textures. This places a computational mechanism, which may be essential for form vision at the earliest stage of cortical processing.
Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription.
Authors: Aronica S M , Kraus W L , Katzenellenbogen B S
Published: May 2006

Estrogenic hormones, believed to exert most of their effects via the direct interaction of their receptors with chromatin, are found to increase cAMP in target breast cancer and uterine cells in culture and in the intact uterus in vivo. Increases in intracellular cAMP are evoked by very low concentrations of estradiol (half maximal at 10 pM) and by other physiologically active estrogens and antiestrogens, but not by an inactive estrogen stereoisomer. These increases in cAMP result from enhanced membrane adenylate cyclase activity by a mechanism that does not involve genomic actions of the hormones (are not blocked by inhibitors of RNA and protein synthesis). The estrogen-stimulated levels of cAMP are sufficient to activate transcription from cAMP response element-containing genes and reporter plasmid constructs. Our findings document a nongenomic action of estrogenic hormones that involves the activation of an important second-messenger signaling system and suggest that estrogen regulation of cAMP may provide an additional mechanism by which this steroid hormone can alter the expression of genes.
The alpha subunit of the Na,K-ATPase specifically and stably associates into oligomers.
Authors: Blanco G , Koster J C , Mercer R W
Published: May 2006

The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit. Both Na,K-ATPase subunits are encoded by multigene families. Several isoforms for the alpha (alpha 1, alpha 2, and alpha 3) and beta (beta 1, beta 2, and beta 3) subunits have been identified. All these isoforms are capable of forming functionally active enzyme. Although there is general agreement that the Na,K-ATPase consists of alpha and beta subunits in equimolar amounts, the quaternary structure of the Na,K-ATPase and its functional significance is unknown. Several studies have demonstrated that the enzyme exists within the plasma membrane as an oligomer of alpha beta dimers. However, because the alpha beta protomer seems to be catalytically competent, the possibility exists that higher oligomers are irrelevant to function. The ability to express different alpha isoforms in insect cells and the availability of isoform-specific antibodies has provided the opportunity to test for the existence of stable and specific associations among alpha subunits. By coexpressing different alpha-subunit isoforms in cultured cells, we demonstrate that the Na,K-ATPase alpha subunits specifically and stably associate into oligomeric complexes. This same association among alpha-subunit isoforms was demonstrated in the native enzyme from rat brain. The interaction between Na,K-ATPase alpha subunits is highly specific. When the Na,K-ATPase alpha subunit is coexpressed with the alpha subunit from the H,K-ATPase, the H,K subunit does not associate with the Na,K subunit. Moreover, expression of the truncated alpha 1T isoform with the full-length alpha subunit demonstrates that the C-terminal portion of the polypeptide is important in the alpha-subunit association. Although these results do not clarify the functional role of alpha alpha associations, they do establish their highly specific nature and suggest that oligomerization of alpha beta protomers may be important to the stability and physiological regulation of the enzyme.
Role of the microtubular system in morphological organization of normal and oncogene-transfected epithelial cells.
Authors: Gloushankova N A , Lyubimova A V , Tint I S , Feder H H , Vasiliev J M , Gelfand I M
Published: May 2006

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.
Ras mediates Src but not epidermal growth factor-receptor tyrosine kinase signaling pathways in GH4 neuroendocrine cells.
Authors: Pickett C A , Gutierrez-Hartmann A
Published: May 2006

p21Ras has been implicated as a critical signaling component in mediating the effects of many growth factor receptor/tyrosine kinases on cell growth and differentiation. However, the precise functional role of Ras in establishing a cell-specific transcriptional response to a ubiquitous growth factor remains unclear. We have utilized a transient cotransfection model system in epidermal growth factor (EGF)-responsive cultured GH4 rat pituitary neuroendocrine cells to investigate the role of Ras in coupling EGF receptor (EGF-R) and v-Src tyrosine kinase signals to the activation of a cell-specific promoter for the rat (r) prolactin (PRL) gene. A significant dose- and time-dependent EGF stimulation of the transfected rPRL promoter was obtained. A similar degree of activation of the rPRL promoter was obtained by cotransfection of a plasmid encoding v-Src. Cotransfection of a construct encoding the dominant-negative Ras, N17Ras, produced almost complete inhibition of v-Src-induced rPRL promoter activity, while EGF-stimulated rPRL promoter activity was unaffected. Similarly, EGF activation of a c-Fos promoter was unaffected by N17Ras, while v-Src activation was blocked. Hence, using transcription regulation as a functional assay, we show that Ras is not required for the EGF-mediated control of the rPRL and c-Fos promoters, whereas Ras is critical in mediating the v-Src effects to these two promoters. These observations emphasize that, despite current biochemical data linking the EGF-R and Ras pathways, the functional significance of such an interaction should be analyzed in a biologically relevant manner and may differ as a function of cell type.
Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.
Authors: Flores-Rozas H , Kelman Z , Dean F B , Pan Z Q , Harper J W , Elledge S J , O'Donnell M , Hurwitz J
Published: May 2006

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.
Receptor for advanced glycation end products (AGEs) has a central role in vessel wall interactions and gene activation in response to circulating AGE proteins.
Authors: Schmidt A M , Hasu M , Popov D , Zhang J H , Chen J , Yan S D , Brett J , Cao R , Kuwabara K , Costache G
Published: May 2006

The extended interaction of aldoses with proteins or lipids results in nonenzymatic glycation and oxidation, ultimately forming AGEs, the presence of which in the plasma and vessel wall is associated with diabetic vascular complications. We show here that AGE albumin in the intravascular space interacts with the vessel wall via binding to an integral membrane protein, receptor for AGE (RAGE), a member of the immunoglobulin superfamily, resulting in clearance from the plasma and induction of interleukin 6 mRNA. Intravenously infused 125I-AGE albumin showed a rapid phase of plasma clearance with deposition in several organs. Rapid removal of 125I-AGE albumin from the plasma was prevented by administration of a soluble, truncated form of RAGE, which blocked binding of 125I-labeled AGE albumin to cultured endothelial cells and mononuclear phagocytes, as well as by pretreatment with anti-RAGE IgG. Ultrastructural studies with AGE albumin-colloidal gold conjugates perfused in situ showed that in murine coronary vasculature this probe was taken up by endothelial plasmalemmal vesicles followed by transport either to the abluminal surface or by accumulation in intracellular vesicular structures reminiscent of endosomes and lysosomes. Consequences of AGE-RAGE interaction included induction of interleukin 6 mRNA expression in mice. These data indicate that RAGE mediates the interaction of AGEs with the vessel wall, both for removal of these glycated proteins from the plasma and for changes in gene expression.
Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat.
Authors: DiGiovanni S R , Nielsen S , Christensen E I , Knepper M A
Published: May 2006

AQP-CD is a vasopressin-regulated water channel expressed exclusively in the renal collecting duct. We have previously shown that AQP-CD is present in the apical plasma membrane and subapical vesicles of collecting duct cells, consistent with membrane-shuttling mechanisms that have been proposed to explain the short-term action of [Arg8] vasopressin (AVP) to regulate apical water permeability. We propose here that AVP may also have long-term actions on the collecting duct to regulate the expression of the AQP-CD water channel. We used immunoblotting, immunohistochemistry, and in vitro perfusion of renal tubules to investigate water channel regulation in collecting ducts of diabetes insipidus (Brattleboro) rats treated with a 5-day infusion of AVP or vehicle. Immunoblotting and immunohistochemistry demonstrated that collecting ducts of vehicle-infused Brattleboro rats had markedly reduced expression of AQP-CD relative to normal rats. In response to AVP infusion there was a nearly 3-fold increase in AQP-CD expression as detected by immunoblotting. Immunocytochemistry demonstrated that the increased expression was predominantly in the apical plasma membrane and subapical vesicles of collecting duct cells. Inner medullary collecting ducts of AVP-infused Brattleboro rats displayed a 3-fold increase in osmotic water permeability relative to vehicle-infused controls, in parallel with the change in AQP-CD expression. Based on these findings, we conclude that (i) long-term infusion of AVP, acting either directly or indirectly, regulates expression of the AQP-CD water channel and (ii) AQP-CD is the predominant AVP-regulated water channel.
Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.
Authors: Ueba T , Nosaka T , Takahashi J A , Shibata F , Florkiewicz R Z , Vogelstein B , Oda Y , Kikuchi H , Hatanaka M
Published: May 2006

Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression.
Convenient separation of high-purity C60 from crude fullerene extract by selective complexation with AlCl3.
Authors: Bucsi I , Aniszfeld R , Shamma T , Prakash G K , Olah G A
Published: May 2006

A purification procedure for C60 from the fullerene extract has been developed using the differential complexation of C60 and C70 with AlCl3 (or its conjugate acid) in CS2 solution. In addition to pure C60 (>99.8%), the procedure also provides C70-enriched fullerene mixture.
Double-stranded cleavage of pBR322 by a diiron complex via a "hydrolytic" mechanism.
Authors: Schnaith L M , Hanson R S , Que L
Published: May 2006

Treatment of plasmid pBR322 with Fe2-(HPTB)(OH)(NO3)4(HPTB = N,N,N',N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopr opane) and H2O2 or O2 and a reductant (dithiothreitol or ascorbate) results in double-stranded cleavage of the plasmid. The linearization of supercoiled pBR322 by this complex is not inhibited by hydroxyl radical scavengers. On the other hand, the linearized pBR322 is efficiently religated by T4 DNA ligase, and the presence of 3'-OH and 5'-OPO3 ends is corroborated by 3'- and 5'-end-labeling studies. These observations indicate that cleavage results from hydrolysis of the DNA-phosphate backbone, which is proposed to occur by nucleophilic attack of the bound peroxide on the phosphodiester. Double-stranded cleavage by the Fe2(HPTB)(OH)(NO3)4/H2O2 adduct preferentially occurs between bp 3489 and 3485 of pBR322.
Characterization of a GTP-binding protein implicated in both memory storage and interorganelle vesicle transport.
Authors: Nelson T J , Yoshioka T , Toyoshima S , Han Y F , Alkon D L
Published: May 2006

The phosphorylation state of cp20, a low molecular weight GTP-binding protein that is a high-affinity substrate for protein kinase C, was previously shown to change after associative conditioning of molluscs and mammals and to induce many of the biophysical and structural modifications that accompany memory retention. Here, cp20 was purified from squid optic lobes and biochemically characterized. A monoclonal antibody prepared against squid cp20 reacted with Hermissenda cp20 and a 20-kDa protein in rabbit hippocampus, while a polyclonal antibody also cross-reacted with Sar1p and ADP-ribosylation factor (ARF). A partial peptide sequence of squid cp20 was 50% identical (23/46 amino acids) with Sar1p, a yeast GTP-binding protein involved in vesicle transport, indicating that cp20 is probably a new member of the ARF family. This classification is consistent with our recent demonstration that cp20 affects retrograde movement of intraaxonal organelles or particles and suggests a possible role for particle traffic between intraneuronal organelles in memory acquisition.
Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties.
Authors: Ballou C E , Ballou L , Ball G
Published: May 2006

Mutagenesis of Schizosaccharomyces pombe cells yielded a strain that made reduced amounts of invertase. A comparison of the O- and N-linked carbohydrate chains of the wild-type and mutant glycoproteins revealed that a single type of alpha 1-->2-linked mannose was missing in the mutant. Analysis of the wild-type galactomannoprotein showed that it contained a heterogeneous small "core" oligosaccharide fraction linked to asparagine with sugar compositions that ranged from Man9(GlcNAc)2- to Gal4Man10(GlcNAc)2-. The galactose units are in terminal positions of a Man10(GlcNAc)2- unit that is similar to the mannoprotein core of Saccharomyces cerevisiae. Attached to this core in a larger oligosaccharide fraction is an alpha 1-->6-linked polymannose chain that is substituted at position 2 with alpha-linked mannose and galactose. The O-linked sugars consist of mannose, alpha 1-->2-linked mannosylmannose and alpha 1-->2-linked galactosylmannose, along with small amounts of tri- and tetrasaccharides. The glycosylation mutant lacks alpha 1-->2-linked mannose on both the O-linked chains and the outer chain of the large N-linked chains, suggesting that it may be defective in regulation of an alpha 1,2-mannosyltransferase that adds mannose to glycoproteins in the Golgi.
Analbuminemia: three cases resulting from different point mutations in the albumin gene.
Authors: Watkins S , Madison J , Galliano M , Minchiotti L , Putnam F W
Published: May 2006

Analbuminemia is a very rare recessive disorder in which subjects have little or no circulating albumin, although albumin is normally the most abundant plasma protein and has many functions. Analbuminemia is caused by a variety of mutations in the albumin gene and is exhibited only by subjects homozygous for the defect. Previously the mutation had been identified at the molecular level in only two human cases; in one case it resulted from an exon-splicing defect, and in the other case it was caused by a nucleotide insertion that caused a frameshift and premature stop codon. In this investigation we identified the mutations in three unrelated subjects from different countries. In each instance a single-nucleotide mutation produced a stop codon, but the mutations occurred at three different sites: (i) in an Italian male a C-->T transition at nt 2368 in the genomic sequence of albumin, (ii) a C-->T transition at nt 4446 for an American female, and (iii) a G-->A transition at nt 7708 in a Canadian male. The size of the albumin fragment that might have been produced for the three cases varied from 31- to 213-amino acid residues, but no evidence for a circulating albumin fragment was obtained. The paradox is that analbuminemia is extremely rare (frequency < 1 x 10(6)); yet the virtual absence of albumin is tolerable despite its multiple functions.
High rate of mismatch extension during reverse transcription in a single round of retrovirus replication.
Authors: Pulsinelli G A , Temin H M
Published: May 2006

We made spleen necrosis virus-based retroviral vectors with mutations at the 3' end of the primer binding site region to observe the effects of terminal mismatches on retroviral replication. These vectors, when compared to a vector with the wild-type primer binding sequence, allowed us to assay the effects of the mutations on the viral titer during a single cycle of replication. The mutant vectors had titers that were comparable to the wild-type vector, indicating that reverse transcriptase has no trouble extending mismatches of as many as 3 bases under normal in vivo conditions. These results confirm and extend previous in vitro studies [Yu, H. & Goodman, M. (1992) J. Biol. Chem. 15, 10888-10896] that showed that such mismatch extension could occur in a cell-free system at high concentrations of incorrect nucleotides and in the absence of correct nucleotides. We now show that mismatch extension can occur during normal retroviral replication in cells and at normal physiological nucleotide concentrations.
Electrostatic distance geometry in a K+ channel vestibule.
Authors: Stocker M , Miller C
Published: May 2006

Many voltage-gated K+ channels carry in the external vestibule a receptor for charybdotoxin, a peptide channel blocker. We use point mutagenesis of both charybdotoxin and a Shaker K+ channel to isolate the electrostatic interaction energy between chosen pairs of residues, one on the channel and one on bound toxin. The results allow estimates of physical distances between such residue pairs and, in combination with the known structure of charybdotoxin, localize specific channel residues in three-dimensional space.
Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.
Authors: Dent P , Sturgill T W
Published: May 2006

The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase. Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown. The ability of preparations of plasma membranes to activate exogenous (His)6-Raf-1 was studied. Plasma membranes of v-Ras-transformed NIH 3T3 cells, but not parental cells, enhanced MAP kinase kinase kinase (MAPKKK) activity dependent on addition of (His)6-Raf-1 and ATP/Mg. Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro. Activation of (His)6-Raf-1 in vitro by membranes was dependent on binding to Ras. Membranes from v-Src-transformed cells also activated (His)6-Raf-1 and synergized with v-Ras membranes. Serum-treatment of NIH 3T3 cells stimulated the ability of membranes to activate (His)6-Raf-1. Activated (His)6-Raf-1 could be recovered on Ni(2+)-agarose, and this methodology was used to demonstrate that activation by membranes was ATP dependent. These findings demonstrate Ras- and ATP-dependent step(s) for Raf-1 activation by plasma membranes in vitro.
Immediate early gene expression associated with the persistence of heterosynaptic long-term depression in the hippocampus.
Authors: Abraham W C , Christie B R , Logan B , Lawlor P , Dragunow M
Published: May 2006

Long-term depression (LTD) of synaptic efficacy is likely to be as important in memory processing as the more well-known long-term potentiation (LTP). The case for LTD serving as a memory mechanism, however, requires that it be shown to persist across days or weeks at least. Here we examined the persistence of heterosynaptic LTD in the medial and lateral perforant path inputs to the dentate gyrus in awake rats and correlated this persistence with the degree of immediate early gene expression as assessed immunohistochemically. Rats were chronically implanted with separate stimulating electrodes in the medial and lateral perforant paths and an extracellular field potential recording electrode in the dentate hilus. After recovery from surgery, either the medial or the lateral perforant path was tetanized with 400-Hz trains, and homosynaptic LTP and heterosynaptic LTD were followed across time. Heterosynaptic LTD was shown to occur readily in awake animals and to persist across days or weeks, depending on the stimulation protocol. The persistence of LTD and LTP was highly correlated within animals. Additional animals, given the same tetanization protocols, showed that the greatest immediate early gene expression occurred following that protocol which consistently gave the longest-lasting LTP and LTD. These data support the proposed role of LTD in memory processing but question whether immediate early genes are important for the persistence of LTP, LTD, or both.
Lateral inhibition mediated by the Drosophila neurogenic gene delta is enhanced by proneural proteins.
Authors: Kunisch M , Haenlin M , Campos-Ortega J A
Published: May 2006

Cells in the neuroectoderm of Drosophila become either neural or epidermal progenitors. A critical threshold concentration of proneural gene products in a given cell causes it to develop as a neuroblast. The proteins encoded by the genes Delta (Dl) and Notch (N) act as the source and the receptor, respectively, of inhibitory signals sent by the neuroblast to neighboring cells that prevent these cells from also adopting the neural fate. We show here that proneural gene products activate transcription of Delta in the neuroectoderm by binding to specific sites in its promoter. This transcriptional activation enhances lateral inhibition and thus helps ensure that cells in the vicinity of prospective neuroblasts will themselves become epidermoblasts.
Learning of the conditioned eye-blink response is impaired by an antisense insulin-like growth factor I oligonucleotide.
Authors: Castro-Alamancos M A , Torres-Aleman I
Published: May 2006

The cerebellum is thought to be critically involved in learning and retention of several types of classically conditioned motor responses. We investigated whether insulin-like growth factor I (IGF-I) may constitute an intercellular mediator of a motor learning task because previous findings indicated that IGF-I from the inferior olive modulates glutamate-induced gamma-aminobutyric acid release by Purkinje cells in the cerebellar cortex. Synaptic plasticity of the Purkinje cell is thought to be instrumental in motor learning. We found that injection of an IGF-I antisense oligonucleotide in the inferior olive elicited a complete inhibition of conditioned eye-blink learning in freely moving rats. This blockage was reversible and recovered when the levels of cerebellar IGF-I returned to normal values. Injection of a sense oligonucleotide did not interfere with the acquisition of the conditioned response. On the other hand, retention of the conditioned response was not impaired by subsequent injection of the IGF-I antisense oligonucleotide, indicating that olivocerebellar IGF-I is essential for the acquisition of the conditioned eye-blink response but is not essential for its retention.
Stimulation of glycogen synthesis by insulin in human erythroleukemia cells requires the synthesis of glycosyl-phosphatidylinositol.
Authors: Lazar D F , Knez J J , Medof M E , Cuatrecasas P , Saltiel A R
Published: May 2006

Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.
Molecular cloning and expression of human leukotriene-C4 synthase.
Authors: Welsch D J , Creely D P , Hauser S D , Mathis K J , Krivi G G , Isakson P C
Published: May 2006

Leukotriene-C4 synthase (LTC4S; EC 2.5.1.37) catalyzes the committed step in the biosynthesis of the peptidoleukotrienes, which are important in the pathogenesis of asthma. Antibodies were generated to a synthetic peptide based on the partial amino acid sequence previously reported for human LTC4S [Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019] and specifically bound detergent-solubilized LTC4S obtained from THP-1 cells, confirming that the published sequence is associated with enzyme activity. Inosine-containing oligonucleotides based on the partial protein sequence were used to isolate a 679-bp cDNA for LTC4S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M(r) = 16,568) that has a calculated pI value of 11.1. The deduced protein sequence is composed predominantly of hydrophobic amino acids; hydropathy analysis predicts three transmembrane domains connected by two hydrophilic loops. Analysis of the deduced sequence identified two potential protein kinase C phosphorylation sites and a potential N-linked glycosylation site. The amino acid sequence for human LTC4S is unique and shows no homology to other glutathione S-transferases. LTC4S was found to be most similar to 5-lipoxygenase activating protein (31% identity, 53% similarity), another protein involved in leukotriene biosynthesis. Active enzyme was expressed in bacterial, insect, and mammalian cells as shown by the biosynthesis of LTC4 in incubation mixtures containing LTA4 and reduced glutathione. The cloning and expression of human LTC4S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis.
Enzymatic completion of mammalian lagging-strand DNA replication.
Authors: Turchi J J , Huang L , Murante R S , Kim Y , Bambara R A
Published: May 2006

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.
Polymorphisms detected by random PCR distinguish between different chromosomal forms of Anopheles gambiae.
Authors: Favia G , Dimopoulos G , della Torre A , Touré Y T , Coluzzi M , Louis C
Published: May 2006

We have applied PCR amplification using random primers to distinguish between incipient species of the malaria vector Anopheles gambiae. Individuals belonging to three chromosomally characterized West African forms of this mosquito, which are important epidemiologically as they differ in vectorial capacity, were sampled both from laboratory stocks and from wild populations collected in three localities. The techniques used allowed for the unambiguous classification of the mosquitoes, providing a tool for rapid and efficient diagnosis, which previously relied on cytological examination of polytene chromosomes.
Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes.
Authors: Yamada Y , Stafforini D M , Imaizumi T , Zimmerman G A , McIntyre T M , Prescott S M
Published: May 2006

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation and atherosclerosis. A specific degradative enzyme found in plasma, PAF acetylhydrolase, plays important roles in various pathophysiological events induced by PAF. Human macrophages and Hep G2 cells secrete PAF acetylhydrolase with characteristics identical to the plasma activity. Other investigators reported that apolipoprotein B may possess phospholipase A2 activity, which suggested that apolipoprotein B might be a zymogen for PAF acetylhydrolase. However, while macrophages express PAF acetylhydrolase activity, we did not detect cDNAs for apolipoprotein B in a cDNA library from these cells, indicating that macrophages do not express this protein. In contrast, Hep G2 cells had high levels of cDNA for apolipoprotein B, as expected. We next injected Xenopus laevis oocytes with poly(A)+ RNA extracted from cultured human macrophages and Hep G2 cells. Twenty-five to 50% of Xenopus oocytes injected with poly(A)+ RNA from macrophages or Hep G2 cells secreted a PAF acetylhydrolase activity (1.0-7.8 nmol/ml per h) that also utilized a synthetic oxidized phospholipid as substrate. The activity secreted by poly(A)+ RNA-injected oocytes associated with lipoproteins and transferred between the particles in a pH-dependent manner, much like the plasma activity. These experiments establish that the properties of the enzyme released from poly(A)+ RNA-injected oocytes are identical to those of the plasma form of PAF acetylhydrolase and that the activity detected is not the expression of a domain in apolipoprotein B.
An evaluation of causes for unreliability of synaptic transmission.
Authors: Allen C , Stevens C F
Published: May 2006

Transmission at individual synaptic contacts on CA1 hippocampal pyramidal neurons has been found to be very unreliable, with greater than half of the arriving presynaptic nerve impulses failing to evoke a postsynaptic response. This conclusion has been reached using the method of minimal stimulation of Schaffer collaterals and whole cell recording in hippocampal slices; with minimal stimulation only one or a few synapses are activated on the target neuron and the behavior of individual synapses can be examined. Four sources for the unreliability of synaptic transmission have been investigated: (i) the fluctuation of axon thresholds at the site of stimulation causing the failure to generate a nerve impulse in the appropriate Schaffer collaterals, (ii) the failure of nerve impulses generated at the site of stimulation to arrive at the synapse because of conduction failures at axon branch points, (iii) an artifactual synaptic unreliability due to performing experiments in vitro at temperatures well below the normal mammalian body temperature, and (iv) transmission failures due to probabilistic release mechanisms at synapses with a very low capacity to release transmitter. We eliminate the first three causes as significant contributions and conclude that probabilistic release mechanisms at low capacity synapses are the main cause of unreliability of synaptic transmission.
Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.
Authors: Petersen H V , Serup P , Leonard J , Michelsen B K , Madsen O D
Published: May 2006

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.
Activation of the human immunodeficiency virus long terminal repeat in THP-1 cells by a staphylococcal extracellular product.
Authors: Klebanoff S J , Kazazi F , Van Voorhis W C , Schlechte K G
Published: May 2006

Staphylococcal strains can release a factor that strongly activates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in THP-1 cells transfected with the HIV-1 LTR-driven luciferase reporter gene (THP-1 LTRluc). The factor is present in the overnight culture fluid and is readily released from the organisms into aqueous medium by vigorous mixing. Staphylococcal extracellular material is a complex mixture of polysaccharide and protein containing peptidoglycan and teichoic acid, released in part by cell wall turnover. The importance of the carbohydrate component is emphasized by concanavalin A (Con A) inhibition of staphylococcal product-induced LTR activation but not of activation by phorbol 12-myristate 13-acetate or tumor necrosis factor. The effect of Con A was decreased or abolished by sugars in the order methyl alpha-D-mannopyranoside > methyl alpha-D-glucopyranoside > mannose > glucose = fructose > N-acetylglucosamine. Wheat germ agglutinin was less inhibitory than Con A; in this instance N-acetylglucosamine decreased inhibition, whereas methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside did not. The induction of luciferase activity in THP-1 LTRluc by the staphylococcal extracellular product also was inhibited by fetal bovine and normal human serum. A comparison of 31 staphylococcal isolates (9 Staphylococcus aureus, 11 Staphylococcus epidermidis, 2 Staphylococcus haemolyticus, 4 Staphylococcus hominis, 2 Staphylococcus capitis, 2 Staphylococcus warneri, 1 Staphylococcus saprophyticus) revealed wide variation in LTR activating activity that did not correlate closely with slime production. Our findings, using induction of luciferase in THP-1 LTRluc as a model for upregulation of HIV infection, raise the possibility that staphylococci, as well as certain other microorganisms, release carbohydrate-containing exopolymers, which can activate the HIV-1 LTR, thus influencing progression of HIV infection.
A catalogue of genes in the cardiovascular system as identified by expressed sequence tags.
Authors: Liew C C , Hwang D M , Fung Y W , Laurenssen C , Cukerman E , Tsui S , Lee C Y
Published: May 2006

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.