bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of angiotensin II AT2 receptors from neonatal rat kidney.
Authors: Ciuffo G M , Heemskerk F M , Saavedra J M
Published: May 2006

Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a nearly homogeneous state after solubilization from neonatal rat kidney membranes with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid. Comparable IC50 values for the soluble extract (0.32 nM) and membranes (0.31 nM) were obtained by competition curves with 125I-labeled CGP42112, a selective AT2 ligand. Binding to AT2 receptors in the soluble extract was not sensitive to dithiothreitol. AT2 receptors were further purified by gel filtration and a CGP42112 Sepharose affinity column. Ang II AT2 receptors were selectively eluted with 5 microM CGP42112 at 4 degrees C, and a single band with an apparent molecular mass of 71 kDa was obtained after SDS/PAGE. Two-dimensional electrophoresis confirmed the purity of the protein and an isoelectric point of 5.3-5.5 was obtained. A highly selective elution of the AT2 receptors from the affinity column was performed with 5 nM 125I-labeled CGP42112 at room temperature after the column was treated with 1 microM losartan in the presence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociation of the radiolabeled protein was insensitive to losartan but was enhanced by CGP42112, PD123177, Ang II, and [Sar1]Ang II. In summary, Ang II AT2 receptors were purified by CGP42112 affinity chromatography and selective elution and retain the pharmacological specificity of particulate receptors.
5-Ethynyluracil (776C85): a potent modulator of the pharmacokinetics and antitumor efficacy of 5-fluorouracil.
Authors: Baccanari D P , Davis S T , Knick V C , Spector T
Published: May 2006

5-Ethynyluracil (5-EU, 776C85) is a mechanism-based irreversible inhibitor of dihydropyrimidine dehydrogenase (EC 1.3.1.2), the rate-determining enzyme in 5-fluorouracil (5-FU) catabolism. In the present study, 5-EU was found to be a potent modulator of 5-FU catabolism in mice and rats. Liver extracts prepared up to 6 hr after a 5-EU dose (2 mg/kg) were > 96% inhibited in their ability to catalyze 5-FU degradation. 5-EU treatment increased the elimination t1/2 and the area under the plasma concentration-time curve of 5-FU. 5-FU oral bioavailability was approximately 100% in rats pretreated with 5-EU. Consequently, 5-EU induced a linear relationship between the area under the plasma concentration-time curve and the oral dose of 5-FU. As expected from the preservation of plasma 5-FU, 5-EU potentiated the antitumor activity and the toxicity of 5-FU in two mouse tumor models (Colon 38 and MOPC-315). However, 5-EU potentiated the antitumor activity to a greater degree and thereby increased the therapeutic index of 5-FU 2- to 4-fold.
Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein.
Authors: Kaiser R , Fernández M R , Parés X , Jörnvall H
Published: May 2006

In contrast to the multiplicity of alcohol dehydrogenase in vertebrates, a class III type of the enzyme [i.e., a glutathione-dependent formaldehyde dehydrogenase; formaldehyde; NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1.] is the only form detectable in appreciable yield in octopus. It is enzymatically and structurally highly similar to the human class III enzyme, with limited overall residue differences (26%) and only a few conservative residue exchanges at the substrate and coenzyme pockets, reflecting "constant" characteristics of this class over wide time periods. It is distinct from the ethanol-active "variable" class I type of the enzyme (i.e., classical liver alcohol dehydrogenase; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The residue conservation of class III is also spaced differently from that of class I but is typical of that of proteins in general, emphasizing that class I, with divergence at three functional segments, is the form with deviating properties. In spite of the conservation in class III, surface charges differ considerably. The apparent absence of a class I enzyme in octopus and the constant nature of the class III enzyme support the concept of a duplicative origin of the class I line from the ancient class III form. Still more distant relationships define further enzyme lines that have subunits with other properties.
Elastic free energy of anisotropic helical ribbons as metastable intermediates in the crystallization of cholesterol.
Authors: Chung D S , Benedek G B , Konikoff F M , Donovan J M
Published: May 2006

We report measurements of the geometrical structure and temporal evolution of metastable helical intermediates in the pathway for cholesterol crystallization in native and model biles. We find that the lecithin component in the bile can dramatically affect the kinetics along this pathway. We also present a theoretical description of these helical intermediates using an elastic free energy appropriate for anisotropic bilayers of tilted chiral amphiphiles, which provides a quantitative description of the observed helical ribbon geometry and insight into the relative free energies of the observed metastable intermediates.
Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.
Authors: Sorkin B C , Jones F S , Cunningham B A , Edelman G M
Published: May 2006

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.
The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans.
Authors: Campbell H D , Schimansky T , Claudianos C , Ozsarac N , Kasprzak A B , Cotsell J N , Young I G , de Couet H G , Miklos G L
Published: May 2006

Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans.
Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.
Authors: Charles I G , Palmer R M , Hickery M S , Bayliss M T , Chubb A P , Hall V S , Moss D W , Moncada S
Published: May 2006

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.
Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c.
Authors: Lu Y , Casimiro D R , Bren K L , Richards J H , Gray H B
Published: May 2006

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.
Translation of the human LINE-1 element, L1Hs.
Authors: McMillan J P , Singer M F
Published: May 2006

Full-length RNA transcribed from the human LINE-1 (L1) element L1 Homo sapiens (L1Hs) has a 900-nt, G+C-rich, 5'-untranslated region (UTR). The 5' UTR is followed by two long open reading frames, ORF1 and ORF2, which are separated from each other by an inter-ORF region of 33 nt that includes two or three in-frame stop codons. We examine here the mechanism(s) by which the translation of L1Hs ORF1 and ORF2 is initiated. A stable hairpin structure (delta G = -74.8 kcal/mol), inserted at nt 661 of the 5' UTR, caused a 3- to 8-fold decrease in the in vitro and in vivo translation of either a lacZ reporter gene for ORF1 or the ORF1 polypeptide product, p40, but translation of a lacZ reporter gene in ORF2 was increased. The results are compatible with a model for ORF1 translation initiation in which the majority of ribosomes scan from a point 5' of nt 661 but suggest that ORF2 is not translated by attached ribosomes that reinitiate after the termination of ORF1 translation. Our data are compatible with a model whereby the translation of L1Hs ORF2 is initiated internally.
The CD58 (LFA-3) binding site is a localized and highly charged surface area on the AGFCC'C" face of the human CD2 adhesion domain.
Authors: Arulanandam A R , Withka J M , Wyss D F , Wagner G , Kister A , Pallai P , Recny M A , Reinherz E L
Published: May 2006

Using site-directed mutagenesis in conjunction with NMR structural data on the adhesion domain of human CD2, we have defined the binding region for CD58. Previous structural studies of rat and human CD2 indicate that this adhesion domain is immunoglobulin-like. Here we report that the CD58 binding site is a well-circumscribed, charged surface area covering approximately 770 A2 on the AGFCC'C" face of the CD2 beta barrel. This site contains beta-strand residues in the carboxyl-terminal half of the F strand (including Lys-82 and Tyr-86), the top of the C strand (Asp-32 and Lys-34), and the C' strand (Gln-46), which are all solvent exposed. In addition, several exposed residues on the FG loop (Gly-90, Lys-91, Asn-92, and Val-93), the CC' loop (Lys-41 and Lys-43), and the C'C" loop (Arg-48 and Lys-51) form this site. In contrast, neither residues on the more peripheral G and C" strands of the same CD2 surface nor residues on B, E, and D strands of the opposite face are involved in CD58 binding. This CD58 binding site is predicted to lie most distal to the T-lymphocyte surface membrane, with ready access to CD58 on the surface of the opposing antigen-presenting cell.
Multiple motifs regulate the B-cell-specific promoter of the B29 gene.
Authors: Omori S A , Wall R
Published: May 2006

The B-cell-specific B29 and mb1 genes code for covalently linked proteins (B29 or Ig beta and mb1 or Ig alpha, respectively) associated with membrane immunoglobulins in the antigen receptor complex on B cells. We have functionally analyzed the upstream region of the B29 gene and have identified a 164-bp region which comprises the minimal promoter responsible for B-cell-specific transcription. Linker scanning mutagenesis of this minimal promoter has established that both the previously identified octamer motif and a DNA motif that binds an unknown protein factor are critical for B29 gene expression in a pre-B-cell and B-cell line. Further mutations showed that binding motifs for Ets, microB/LyF1, and Sp1 also significantly contributed to the overall activity of the minimal B29 promoter. However, the relative contribution of certain motifs to promoter activity was different in a pre-B versus a B-cell line. The microB/LyF1 motif was necessary for full promoter activity in the pre-B cells but was not required in the B cells.
Conversion of G-protein specificity of insulin-like growth factor II/mannose 6-phosphate receptor by exchanging of a short region with beta-adrenergic receptor.
Authors: Takahashi K , Murayama Y , Okamoto T , Yokota T , Ikezu T , Takahashi S , Giambarella U , Ogata E , Nishimoto I
Published: May 2006

The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.
The minimal self-sufficient element in a murine G+C-rich promoter is a large element with imperfect dyad symmetry.
Authors: Ackerman S L , Minden A G , Yeung C Y
Published: May 2006

The murine adenosine deaminase gene has a structurally archetypal TATAA-box-deficient G+C-rich promoter. The three Sp1 binding sites of the promoter are neither necessary nor sufficient for promoter function. Minimal basal promoter activity resides within a 48-bp element downstream of the Sp1 binding sites. This element shows an imperfect dyad symmetry around the promoter's major transcriptional initiation site and contains at least two nuclear protein binding sites. The distinctive sequence characteristics and nuclear protein binding locations of this element led us to propose a model for how such promoters may function.
Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits.
Authors: Keaney J F , Gaziano J M , Xu A , Frei B , Curran-Celentano J , Shwaery G T , Loscalzo J , Vita J A
Published: May 2006

Recent evidence suggests that dietary therapy with lipid-soluble antioxidants may be beneficial for patients with atherosclerotic vascular disease but the potential mechanism(s) for these observations remain obscure. Abnormalities in endothelium-dependent control of vascular tone develop early in the course of atherosclerosis and may result from oxidative modification of low density lipoproteins. We examined the role of dietary antioxidants in preserving normal endothelial cell vasodilator function in cholesterol-fed rabbits with particular attention to possible effects on serum lipoproteins, low density lipoprotein oxidation, and atherogenesis. Male New Zealand White rabbits were fed diets containing no additive (controls), 1% cholesterol (cholesterol group), or 1% cholesterol chow supplemented with either beta-carotene (0.6 g/kg of chow) or alpha-tocopherol (1000 international units/kg of chow) for a 28-day period. After dietary therapy, thoracic aortae were harvested for assay of vascular function and for pathologic examination and tissue antioxidant levels. Compared to controls, acetylcholine- and A23187-mediated endothelium-dependent relaxations were significantly impaired in vessels from the cholesterol group (P < 0.001), whereas vessels from animals treated with beta-carotene or alpha-tocopherol demonstrated normal endothelium-dependent arterial relaxation. Preservation of endothelial function was associated with vascular incorporation of alpha-tocopherol and beta-carotene but was unrelated to plasma lipoprotein levels, smooth muscle cell function, or the extent of atherosclerosis. Increased low density lipoprotein resistance to ex vivo copper-mediated oxidation was observed only in the alpha-tocopherol group. Our results suggest that dietary antioxidants may benefit patients with atherosclerosis by preserving endothelial vasodilator function through a mechanism related to vascular tissue antioxidant content and not reflected by assay of low density lipoprotein resistance to ex vivo oxidation.
Interleukin 6 inhibits mouse placental lactogen II but not mouse placental lactogen I secretion in vitro.
Authors: Yamaguchi M , Ogren L , Southard J N , Kurachi H , Miyake A , Talamantes F
Published: May 2006

The mouse placenta produces several polypeptides belonging to the prolactin-growth hormone gene family, including mouse placental lactogen (mPL) I and mPL-II. The present study was undertaken to determine whether the secretion of mPL-I and mPL-II is regulated by interleukin 6 (IL-6), which is present in the placenta and has previously been reported to stimulate the secretion of pituitary members of this gene family. Effects of human and mouse IL-6 on mPL-I and mPL-II secretion were examined in primary cultures of placental cells from days 7, 9, and 12 of pregnancy. IL-6 caused a dose-dependent reduction in the mPL-II concentration in the medium of cells from days 9 and 12 of pregnancy but did not affect the mPL-II concentration in the medium of cells from day 7 of pregnancy or the mPL-I concentration in the medium of cells from days 7 or 9 of pregnancy. The lowest concentration of human IL-6 that significantly inhibited mPL-II secretion was 250 pM. The effect of IL-6 on the mPL-II concentration in the medium was due primarily to inhibition of mPL-II synthesis, which resulted at least partly from a decrease in the steady-state level of mPL-II mRNA. These data raise the possibility that IL-6 may regulate mPL-II production after midpregnancy in vivo.
The bovine papillomavirus origin of replication requires a binding site for the E2 transcriptional activator.
Authors: Ustav E , Ustav M , Szymanski P , Stenlund A
Published: May 2006

The bovine papillomavirus type I transcriptional activator E2 is essential for replication of bovine papillomavirus DNA, yet most of the high-affinity binding sites for E2 are dispensable. Here we demonstrate an absolute requirement for a binding site for the E2 polypeptide as a cis-acting replication element, establishing that site-specific binding of E2 to the origin is a prerequisite for bovine papillomavirus replication in vivo. The position and distance of the E2 binding site relative to the other origin of replication components are flexible, but function at a distance requires high-affinity E2 binding sites. Thus, low-affinity binding sites function only when located close to the origin of replication, while activity at greater distances requires multimerized high-affinity E2 binding sites. The requirement for E2, although different in some respects, shows distinct similarities to what has been termed replication enhancers and may provide insight into the function of this class of DNA replication element.
Chemically bonded stationary phases that use synthetic hosts containing aromatic binding clefts: HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons.
Authors: Zimmerman S C , Saionz K W , Zeng Z
Published: May 2006

The synthesis of hosts with improved binding affinities for nitroaromatic guests is described. Association constants for several host-guest complexes were measured in chloroform solution and ranged over three orders of magnitude. Two hosts were covalently linked to silica gel to produce chemically bonded stationary phases for HPLC. The use of these phases for HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons is discussed.
Recognition and catalysis in nucleic acid chemistry.
Authors: Breslow R , Xu R
Published: May 2006

The enzyme ribonuclease A catalyzes the cleavage of RNA, using the imidazole groups of histidine-12 and histidine-119 as its principal catalytic groups. Model studies show that RNA can be cleaved by imidazole buffer itself and that, as in the enzyme, a bell-shaped pH vs. rate profile is seen. This indicates that one imidazole functions as a base, while the other, as the imidazolium ion, functions as an acid. However, in contrast to the enzymatic case, the simple model uses the imidazoles in sequential, rather than simultaneous, bifunctional catalysis. Mechanistic studies on this reaction and on the reactions of simple dinucleotides catalyzed by imidazole and other buffers establish the details of the process. The results let us propose a mechanism for the enzymatic process different from the standard one; they also stimulated us to design an improved mimic of the enzyme that uses a mechanism like that proposed for the enzyme. Critical to the mechanistic studies is observation of the rearrangement of normal 3',5'' RNA nucleotides to the 2',5'' isomers. This led us to investigate the properties of DNA isomers in which a 2',5'' link also replaces the normal 3',5'' one. The results indicate that poor base stacking in a double helix with such links makes them less suitable as genetic units.
Identification of two distinct properties of class II major histocompatibility complex-associated peptides.
Authors: Nelson C A , Petzold S J , Unanue E R
Published: May 2006

We have examined the interactions of various peptides with the mouse class II major histocompatibility complex molecule I-Ak. The peptides were derived from the model protein hen egg white lysozyme (HEL). The immunodominant peptide of HEL is a 10-mer, residues 52-61. Our previous work established that this sequence contains the key residues for binding and presentation to T cells. Now we show that the binding of this 10-mer sequence resulted in complexes of I-Ak and peptide that, in SDS/PAGE (without boiling the protein), rapidly dissociated from the component alpha and beta chains. The binding interactions were studied in vitro, by incubating purified I-Ak and radiolabeled peptide, or ex vivo, by using antigen-presenting cells incubated with peptides. Peptides with additional residues at either the amino or carboxyl terminus behaved dramatically differently. Complexes of I-Ak with the longer peptides were stable to SDS/PAGE. Very few amino acid additions result in the change from unstable to stable complexes. The important issue here is that when cultured with HEL, antigen-presenting cells selected the HEL peptides containing the 52-61 sequences that favored stability [Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. (1992) Proc. Natl., Acad. Sci. USA 89, 7380-7383]. Also, from other studies, such sequences correlate with a high immunogenicity of the peptide. We conclude that there are structural features of peptides that change the stability of the class II molecule and that are independent of the "core" peptide seen by the T cells.
Chronic growth stimulation of human adult melanocytes by inflammatory mediators in vitro: implications for nevus formation and initial steps in melanocyte oncogenesis.
Authors: Medrano E E , Farooqui J Z , Boissy R E , Boissy Y L , Akadiri B , Nordlund J J
Published: May 2006

In the human epidermis, melanocytes are distributed at a distance from each other. In contrast, melanocytes in nevi, which are considered benign neoplasms of melanocytes, are grouped in nests. Although still not well defined, environmental factors are thought to play an important role in the development of nevi. We found that chronic growth stimulation by leukotriene C4, a compound found in increased amounts in inflamed skin, induced pleiotropic modifications in the normal melanocyte phenotype. These changes include loss of contact inhibition and formation of structures resembling tumor spheroids. In parallel with these changes, there was a constitutive expression of Fos protein. Switching these cultures to medium supplemented with phorbol ester sustained growth with reversion of the altered phenotype. In contrast, a cAMP stimulator, cholera toxin, induced features of terminal differentiation. Our findings suggest a role for inflammatory mediators in human epidermal melanocytes. This observation provides insight into melanocyte growth alterations which may have relevance in early stages of melanocyte oncogenesis.
Functional expression of the alpha 2 and alpha 3 isoforms of the Na,K-ATPase in baculovirus-infected insect cells.
Authors: Blanco G , Xie Z J , Mercer R W
Published: May 2006

Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha 2, alpha 3, and beta 1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha 2 and alpha 3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha 2 or alpha 3 with beta 1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-32P]ATP or 32Pi to identify the phosphorylated intermediates of the alpha 2 and alpha 3 isoforms. [3H]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha 3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.
Peptide analogues alter the progression of premalignant lesions, as measured by Photofrin fluorescence.
Authors: Liebow C , Crean D H , Schally A V , Mang T S
Published: May 2006

Somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 were infused at 2 micrograms per day via miniosmotic pumps implanted s.c. in hamsters with premalignant disease to examine the effect of these peptides on cancer promotion and progression. These analogues have been shown to inhibit growth of certain tumors, especially those that overexpress tyrosine kinase activity. Progression of premalignant lesions initiated by applying 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) to the hamster buccal cheek pouch was measured by Photofrin-induced fluorescence 24 hr after injecting the porphyrin (1.0 mg/kg) by using in vivo fluorescence photometry. This method of monitoring progression was reaffirmed by the observations that fluorescence increased significantly as compared with controls in lesions receiving 4 additional weeks of continuous promotion by DMBA application (P < 0.01 in two independent trials) and in lesions receiving transient promotion by laser incision (P < 0.01 and < 0.05 at the same time in the two trials). Twelve weeks after treatment, fluorescence had decreased significantly among animals treated for 2 weeks with RC-3095 (control, 0.53 +/- 0.03 V vs. RC-3095, 0.28 +/- 0.03 V; P < 0.0005) or with RC-160 (control, 0.85 +/- 0.03 V vs. RC-160, 0.24 +/- 0.03 V; P < 0.0001). These data were obtained 20 weeks after DMBA initiation. Thus, treatment with RC-160 and RC-3095 decreased the progression, measured by fluorescence, compared with control animals. In addition, there was also an absolute continuous decrease in fluorescence for the 22 weeks after the cessation of RC-160 treatment. That the changes in tumor progression produced by RC-160 extended beyond the treatment period supports the hypothesis that the changes were irreversible. Histopathological analysis revealed normal tissue and/or mild-moderate dysplasia in hamster buccal mucosa treated with the RC-160 (an improvement compared to pretreatment), whereas 40% of the animals receiving no treatment after DMBA initiation developed invasive squamous cell carcinomas after 20 weeks. These results show that the antagonists of bombesin/gastrin-releasing peptide can delay the development of malignancies and the agonists of somatostatin can potentially reverse this development.
Expression of insulin-like growth factor I gene in osmoregulatory organs during seawater adaptation of the salmonid fish: possible mode of osmoregulatory action of growth hormone.
Authors: Sakamoto T , Hirano T
Published: May 2006

Growth hormone has been shown to contribute to seawater adaptation of salmonid fishes. The growth influence of growth hormone is mediated largely by hepatic production of insulin-like growth factor I (IGF-I). To study the growth hormone-IGF-I axis in osmoregulation, we measured IGF-I mRNA in the liver, gill, and body kidney from rainbow trout by Northern analysis. The levels of IGF-I mRNA in all tissues increased significantly after injection of growth hormone. Transfer of trout from fresh water to 80% seawater evoked an increase in plasma growth hormone after 1 day. IGF-I mRNA was not altered significantly in the liver, but it was increased in the gill and body kidney after 1 and 8 days, respectively. These observations indicate that the IGF-I gene is expressed differently among these organs during seawater adaptation. Growth hormone may stimulate hypoosmoregulatory ability by inducing local expression of IGF-I in osmoregulatory organs, although the possibility that IGF-I expression might occur in part independently of growth hormone during seawater adaptation cannot be excluded.
Linkage of Niemann-Pick disease type C to human chromosome 18.
Authors: Carstea E D , Polymeropoulos M H , Parker C C , Detera-Wadleigh S D , O'Neill R R , Patterson M C , Goldin E , Xiao H , Straub R E , Vanier M T
Published: May 2006

We analyzed the involvement of chromosome 18 in Niemann-Pick disease type C (NPC), an autosomal recessive cholesterol-processing disorder. Within affected offspring, the chromosome 18 parental contributions were identified by using allele-specific microsatellite markers. Significant linkage of NPC to an 18p genomic marker, D18S40, was indicated by a two-point lod score of 3.84. Analysis of meiotic chromosomal breakpoint patterns among the affected individuals indicated that the NPC gene is pericentromerically localized on human chromosome 18.
Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.
Authors: Younkin D P , Tang C M , Hardy M , Reddy U R , Shi Q Y , Pleasure S J , Lee V M , Pleasure D
Published: May 2006

Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.
Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system.
Authors: Clatterbuck R E , Price D L , Koliatsos V E
Published: May 2006

The neurocytokine ciliary neurotrophic factor (CNTF) was described originally as an activity that supports the survival of neurons of the chicken ciliary ganglia in vitro. The widespread expression of CNTF and its principal binding protein, CNTF receptor alpha, in the central and peripheral nervous systems suggests a broader trophic role for this peptide. In the present study, we report that CNTF prevents axotomy-induced cell death of neurons in the anteroventral and anterodorsal thalamic nuclei of the adult rat. Using the polymerase chain reaction, we also demonstrate the presence of CNTF and CNTF receptor alpha mRNA in these same thalamic nuclei. The coincidence of CNTF and its receptor in a population of neurons responding to the factor suggests a paracrine function for CNTF. The present findings establish that CNTF has significant effects on neurons of the central nervous system in vivo and demonstrate that neurocytokines can prevent cell death in the adult central nervous system.
Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation.
Authors: Patriotis C , Makris A , Bear S E , Tsichlis P N
Published: May 2006

The Tpl-2 locus, cloned by provirus tagging from one of three sublines of the Moloney leukemia virus-induced rat thymoma 2769, defines a gene encoding a protein kinase associated with progression in 22.5% of the tumors. Tpl-2 is expressed primarily in spleen, thymus, liver, and lung. Provirus integration occurs in the last intron of the gene, leading to the expression of a truncated mRNA that terminates in the proviral long terminal repeat and encodes a protein with an altered C-terminal domain. Strong evidence that this genetic change confers growth advantage to affected cell clones was provided by the finding that, during cultivation of all three sublines derived from tumor 2769, cells were selected that harbored independent provirus insertions in the Tpl-2 locus. Exposure of normal rat spleen cells to Con A induces the expression of enhanced levels of Tpl-2 within the first 60 min from the time of exposure suggesting that, in normal splenocytes, Tpl-2 may be involved in the transition from a quiescent to the G1 phase of the cell cycle.
SpoIIAB is an anti-sigma factor that binds to and inhibits transcription by regulatory protein sigma F from Bacillus subtilis.
Authors: Duncan L , Losick R
Published: May 2006

The sigma F factor is a regulatory protein that is responsible for directing gene expression in the forespore compartment of developing cells of the spore-forming soil bacterium Bacillus subtilis. The sigma F factor is encoded by the promoter-distal member of sporulation operon spoIIA, which consists of cistrons called spoIIAA, spoIIAB, and spoIIAC. Genetic evidence indicates that the activity of sigma F is negatively regulated by the product (SpoIIAB) of the spoIIAB cistron. We now report that SpoIIAB is capable of binding to sigma F and inhibiting its capacity to direct transcription by core RNA polymerase from the promoter for a forespore-expressed gene. SpoIIAB is an anti-sigma factor that may be directly involved in the compartmentalization of sigma F-directed gene expression.
Ontogeny of anti-human immunodeficiency virus (HIV) antibody production in HIV-1-infected infants.
Authors: Pollack H , Zhan M X , Ilmet-Moore T , Ajuang-Simbiri K , Krasinski K , Borkowsky W
Published: May 2006

The early serologic response of infants to infection with human immunodeficiency virus type 1 (HIV-1) is normally obscured by the presence of transplacentally acquired maternal HIV antibody. By measuring HIV antibody produced in vitro by lymphocytes isolated from peripheral blood of infants and children of HIV-1-infected mothers, we have been able to study the natural acquisition of humoral immunity to perinatal HIV-1 infection. One hundred ninety-seven infants of HIV-1-infected women were studied prospectively and longitudinally from birth. In the neonatal period, infected infants produced only small amounts of HIV-specific IgG antibodies to a restricted number of antigens. The amount of immunoglobulin to HIV-1 and the number of HIV-1 antigens recognized increased with age. After 6 months of life 85% of infected infants made detectable antibody to two or more viral proteins. Antibody to gp160 appeared first and was the most frequently found at all ages, followed by antibody to the envelope proteins gp120 and gp41. The amount of HIV antibody produced correlated positively with the percentage of CD4+ T lymphocytes in peripheral blood. This assay provides a method of studying the immunogenicity of vaccines against HIV-1 in HIV-1-infected infants and of assessing the effect of early therapeutic interventions on the humoral response to HIV-1.
Immunoglobulin heavy chain constant region determines the pathogenicity and the antigen-binding activity of rheumatoid factor.
Authors: Fulpius T , Spertini F , Reininger L , Izui S
Published: May 2006

An IgG3 monoclonal antibody, 6-19, derived from unmanipulated MRL/MpJ-lpr/lpr mice, exhibiting cryoglobulin and anti-IgG2a rheumatoid factor activities, induces skin leukocytoclastic vasculitis and glomerulonephritis when injected into normal mice. To determine the role of the gamma 3 heavy chain constant region in the generation of cryoglobulins and associated tissue lesions, we have established an IgG1 class switch variant, clone SS2F8, from the 6-19 hybridoma by sequential sublining. Here we report that the SS2F8 monoclonal antibody, which loses the cryoglobulin activity but retains the rheumatoid factor activity, fails to generate skin and glomerular lesions. The lack of pathogenicity of the IgG1 SS2F8 switch variant is not due to mutations in variable regions, since nucleotide sequence analysis shows no differences between both clones. In addition, we have observed that the IgG1 SS2F8 switch variant exhibits < 10% of the rheumatoid factor activity, as compared with the IgG3 6-19 monoclonal antibody, suggesting that the self-associating property of the gamma 3 isotype promotes antibody-binding activity. The present study indicates that the cryoglobulin activity associated with the gamma 3 isotype is critically involved in the pathogenicity of 6-19 anti-IgG2a rheumatoid factor monoclonal antibody and highlights the pathogenic relevance of autoantibodies of the IgG3 subclass in murine systemic lupus erythematosus.
Bordetella pertussis tracheal cytotoxin and other muramyl peptides: distinct structure-activity relationships for respiratory epithelial cytopathology.
Authors: Luker K E , Collier J L , Kolodziej E W , Marshall G R , Goldman W E
Published: May 2006

Tracheal cytotoxin (TCT) is a disaccharide-tetrapeptide released by Bordetella pertussis, the causative agent of pertussis (whooping cough). We have previously determined the structure of TCT to be GlcNAc-1,6-anhydro-MurNAc-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala, where MurNAc = N-acetylmuramic acid and A2pm = diaminopimelic acid. Purified TCT reproduces the respiratory cytopathology observed during pertussis, including ciliostasis and extrusion of ciliated cells. We have tested structural analogs of TCT for their ability to reproduce native TCT toxicity in explanted hamster tracheal tissue and hamster trachea epithelial (HTE) cell cultures. Other investigators have evaluated many of these analogs, which are muramyl or desmuramyl peptides, for muramyl peptide activities such as immunopotentiation, induction of slow-wave sleep, and pyrogenicity. Four desmuramyl peptides were produced in our laboratory from B. pertussis peptidoglycan or by chemical synthesis, including unusual peptides containing alpha-aminopimelic acid in place of A2pm. Based on the relative ability of compounds to inhibit DNA synthesis in HTE cells, truncated analogs lacking A2pm entirely or lacking only the side-chain amine or carboxyl group of A2pm were less active than TCT by a factor of at least 1000. All active analogs included a native or near-native peptide moiety, independent of the presence, absence, or substitution of the sugar moiety. We conclude that the structural requirements for TCT toxicity differ considerably from those for most other muramyl peptide activities, in that the disaccharide moiety is irrelevant for toxicity and both the free amino and carboxyl groups of the A2pm side chain are required for activity.
The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer.
Authors: Cockerill P N , Shannon M F , Bert A G , Ryan G R , Vadas M A
Published: May 2006

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-cell line to identify additional regulatory elements. We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an AP1-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.
Insulin receptor isotype expression correlates with risk of non-insulin-dependent diabetes.
Authors: Mosthaf L , Eriksson J , Häring H U , Groop L , Widen E , Ullrich A
Published: May 2006

Skeletal muscle insulin resistance plays a pivotal role in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM), as individuals with this defect are at increased risk of developing the disease later in life. To assess whether the abnormal expression of the structurally distinct human insulin receptor isoforms, HIR-A and HIR-B, which has been found in skeletal muscle of NIDDM patients, is a feature of a prediabetic state, skeletal muscle biopsies from nondiabetic individuals ranging from high insulin sensitivity to insulin resistance were examined. Polymerase chain reaction analysis of mRNA from muscle biopsies detected exclusive or predominant expression of HIR-A in 13 patients with normal insulin sensitivity. In contrast, 7 subjects with various degrees of insulin resistance exhibited abnormally increased HIR-B RNA expression. This association suggests the abnormal expression of receptor isoforms as a characteristic of the prediabetic state and supports the notion of a connection of this aberration with the pathogenesis of NIDDM. Changes in HIR-A/B expression in the skeletal muscle may thus provide a prognostic criterion for the development of NIDDM.