bluuebunny/crossref_metadata_2025 huggingface dataset:
Model of the active site in the blue oxidases based on the ceruloplasmin-plastocyanin homology.
Authors: Rydén L
Published: May 2006

Available sequence information (a total of 650 residues out of about 1,050) of human ceruloplasmin, a blue copper-containing oxidase, has been examined for internal homologies and relationships to other blue proteins. The peptide chain has an evident 3-fold repeat of about 340 residues, and weak evidence for a 6-fold repeat of 170 residues exists. When another method was used to compare the longer sequence with the sequences of small blue proteins, azurins and plastocyanins, a 109-residue-long sequence at the COOH terminus of ceruloplasmin was found to be homologous to the plastocyanins. The alignment obtained was used to construct, on a graphic display, a three-dimensional model of this part of ceruloplasmin by using the coordinates for popular plastocyanin. Deletions and insertions could be accommodated in turns and kinks in the essentially eight-stranded pleated sheet molecule wherein each of the hydrophobic core residues was conserved or conservatively replaced. Eight of the 12 histidine side chains were clustered at or close to the binding site for the blue (type 1) copper. On the assumption that these are copper ligands, a model for the active site of ceruloplasmin containing four copper ions could be constructed in a manner consistent with known spectroscopic and kinetic data. In particular, two of the coppers are close enough (3 A) to form a binuclear center. The positions of the two additional coppers (the fifth and the sixth) in ceruloplasmin are suggested on the basis of the internal homologies.
Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.
Authors: Mitra S , Bersohn R
Published: May 2006

On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochemistry 15, 775-786] postulated a slowly attained (approximately equal to 0.1 sec) conformational equilibrium between two forms of reduced azurin from the bacterium Pseudomonas aeruginosa, one form being faster in electron transfer. NMR investigations have shown that at pH 7 there are two forms of reduced azurin exchanging slowly with each other, differing in the presence or absence of a proton on the imidazole side chain of histidine-35. Rosen et al. [Rosen, P., Segal, M. & Pecht, I. (1981) Eur. J. Biochem. 120, 339-344] observed that the azurin from the bacterium Alcaligenes faecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(II) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH less than 5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure of P. aeruginosa azurin shows that at pH 7 the imidazole side chain of histidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that of histidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.
Direct evidence against a role of ATP as the nonadrenergic, noncholinergic inhibitory neurotransmitter in guinea pig tenia coli.
Authors: Westfall D P , Hogaboom G K , Colby J , O'Donnell J P , Fedan J S
Published: May 2006

Electrical field stimulation of the isolated guinea pig tenia coli in the presence of a muscarinic receptor antagonist (atropine) and an adrenergic neuron blocker (guanethidine) produces relaxation. A large amount of indirect evidence has suggested that the neurotransmitter that is released from these nonadrenergic, noncholinergic inhibitory neurons is ATP or a related nucleotide, and the nerves have been termed "purinergic." A photoaffinity analog of ATP, arylazido aminopropionyl ATP, which produces a specific pharmacological antagonism of P2 purinergic receptors in isolated guinea pig vas deferens and urinary bladder, was utilized in the present study to evaluate directly whether ATP is the nonadrenergic, noncholinergic inhibitory neurotransmitter in tenia coli. By blocking postjunctional P2 receptors, arylazido aminopropionyl ATP produced a pronounced antagonism of relaxations induced by exogenously added ATP. Responses produced by ADP, AMP, and adenosine also were antagonized by arylazido aminopropionyl ATP, but to a lesser extent. Inhibitory responses to isoproterenol were not antagonized. Under these conditions of established, specific P2-receptor blockade of responses to exogenously added ATP, relaxations induced by field stimulation of intrinsic inhibitory nerves in the presence of atropine (1 microM) and guanethidine (1 microM) were not antagonized. Though these results provide no indication of the actual substance involved, they suggest strongly that the nonadrenergic, noncholinergic inhibitory neurotransmitter in the guinea pig tenia coli is not ATP.
Preparation and characterization of a pentaammineruthenium(III) derivative of horse heart ferricytochrome c.
Authors: Yocom K M , Shelton J B , Shelton J R , Schroeder W A , Worosila G , Isied S S , Bordignon E , Gray H B
Published: May 2006

A stable complex between pentaammineruthenium(III) and histidine-33 in horse heart ferricytochrome c is formed in the reaction between aquopentaammineruthenium(II) and the protein at pH 7. HPLC of the tryptic hydrolysate of the modified protein was employed to identify the pentaammineruthenium binding site. Spectroscopic measurements show that the integrity of the native structure in the vicinity of the heme c group is maintained in the ruthenium-modified protein. The reduction potentials are: heme c (Fe3+/2+), 0.26 V; Ru(NH3)5(His-33)3+/2+, 0.15 V (vs. normal hydrogen electrode).
Rat pancreatic kallikrein mRNA: nucleotide sequence and amino acid sequence of the encoded preproenzyme.
Authors: Swift G H , Dagorn J C , Ashley P L , Cummings S W , MacDonald R J
Published: May 2006

We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-noncoding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the gamma subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.
A transposable element from Halobacterium halobium which inactivates the bacteriorhodopsin gene.
Authors: Simsek M , DasSarma S , RajBhandary U L , Khorana H G
Published: May 2006

We describe the characterization of a transposable element from an archaebacterium. The bacteriorhodopsin genes from the wild-type and two mutant Halobacterium halobium strains have been cloned as BamHI fragments in pBR322. The cloned DNA fragments from the two mutants both contain a 1.1-kilobase-pair insertion sequence (ISH1) near the NH2 terminus of the bacteriorhodopsin coding sequence. ISH1 is present in the two mutants in an identical palindromic site but in opposite orientations. The complete sequence of ISH1 has been determined; it is 1,118 nucleotides long, it has 8-base-pair interrupted inverted repeats at the ends, and it duplicates an 8-base-pair (A-G-T-T-A-T-T-G) target sequence upon insertion. As for most eukaryotic and some prokaryotic transposable elements, the sequence of the ISH1 begins with T-G and ends in C-A. ISH1 contains an open reading frame 810 nucleotides long and codes for an RNA approximately 900 nucleotides long. The copy number of ISH1 ranges from one to five or more in different H. halobium strains. In at least one of the strains, one copy of ISH1 is present also on a plasmid DNA.
Transient kinetics of the rapid shape change of unstirred human blood platelets stimulated with ADP.
Authors: Deranleau D A , Dubler D , Rothen C , Lüscher E F
Published: May 2006

Unstirred (isotropic) suspensions of human blood platelets stimulated with ADP in a stopped-flow laser turbidimeter exhibit a distinct extinction maximum during the course of the classical rapid conversion of initially smooth flat discoid cells to smaller-body spiny spheres. This implies the existence of a transient intermediate having a larger average light scattering cross section (extinction coefficient) than either the disc or the spiny sphere. Monophasic extinction increases reaching the same final value were observed when either discoid or spiny sphere platelets were converted to smooth spheres by treatment with chlorpromazine, and sphering of discoid cells was accompanied by a larger total extinction change than the retraction of pseudopods by already spherical cells. These and other results suggest that the ADP-induced transient state represents platelets that are approximately as "spherical" as the irregular spiny sphere but lack the characteristic long pseudopods and as a consequence are larger bodied. Fitting the ADP progress curves to the series reaction A leads to B leads to C by means of the light scattering equivalent of the Beer-Lambert law yielded scattering cross sections that are consistent with this explanation. The rate constants for the two reaction steps were identical, indicating that ADP activation corresponds to a continuous random (Poisson) process with successive apparent states "disc," "sphere," and "spiny sphere," whose individual probabilities are determined by a single rate-limiting step.
Gonadotropin-releasing hormone stimulation of luteinizing hormone release: A ligand-receptor-effector model.
Authors: Blum J J , Conn P M
Published: May 2006

A divalent antibody conjugate of a pure antagonist of gonadotropin-releasing hormone (GnRH) behaved as an agonist--i.e., released luteinizing hormone (LH) from pituitary cultures. Release was measured over a wide range of conjugate concentrations; it rose to a maximum of 66% of the LH released by the optimal concentration of GnRH and declined to basal levels at very high concentrations. This behavior was modeled on the assumption that the antibody conjugate, A, can react with a receptor, R, to form a complex, A . R2. This dimer then can react with a quiescent effector, E (e.g., a closed Ca2+ ion channel), to form A . R2 . E, which contains activated effector and leads to cellular responses. The equilibrium equations governing the behavior of this model were derived, solved, and found to yield a good fit to the experimental data. Consideration of our data in this model system, and of other available data describing the behavior of ligands in other cells, suggests that the present model may be of wide applicability.
Polyadenylylated RNA complementary to a mouse retrovirus-like multigene family is rapidly and specifically induced by epidermal growth factor stimulation of quiescent cells.
Authors: Foster D N , Schmidt L J , Hodgson C P , Moses H L , Getz M J
Published: May 2006

Complementary DNA probes prepared from total polysomal poly(A)+RNA populations were used to identify clones of mouse DNA containing sequences whose expression is specifically enhanced after epidermal growth factor (EGF) stimulation of quiescent mouse embryo cells in culture. Three such clones were isolated and used to study changes in the levels of clone-specific poly(A)+RNA in the polysomes of cells after mitogenic stimulation by EGF. RNA complementary to sequences present in these clones increased approximately equal to 10-fold as a fraction of the total poly(A)+RNA by 6 hr after stimulation. All three clones were found by hybridization criteria to contain sequences related to the class of mouse retrovirus or transposon-like elements termed VL30. These VL30-related sequences were further found to be complementary to EGF-inducible poly(A)+RNAs and enhanced expression was detectable as early as 1 hr after EGF stimulation. In contrast, nine additional clones, including an AKR-type murine leukemia provirus DNA clone, contained no detectable VL30 sequence elements and were complementary to poly(A)+RNA species whose relative concentration was essentially constant in quiescent and EGF-stimulated cells. Therefore, VL30 sequence elements appear distinct in that they encompass members whose expression is specifically regulated in response to a defined peptide growth factor.
Mucolipidosis III is genetically heterogeneous.
Authors: Honey N K , Mueller O T , Little L E , Miller A L , Shows T B
Published: May 2006

Mucolipidosis III (ML III), or pseudo-Hurler polydystrophy, is an inherited childhood disorder characterized biochemically by low activities and abnormal electrophoretic patterns of multiple lysosomal enzymes in fibroblasts. The primary deficiency of ML III has been proposed to be in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. However, variation in this enzyme and in other biochemical properties of different ML III lines has been observed. Therefore, we investigated genetic heterogeneity within the disorder by complementation analysis. Heterokaryon cell fractions were generated by fusing together ML III fibroblast lines. When pairs of cells complemented, correction of lysosomal enzyme activities and electrophoretic patterns was observed. Twelve fibroblast lines from 10 sibships were analyzed and three distinct complementation groups were characterized. One complementation group represents the classical ML III disorder. A single cell line identifies a second complementation group. The cell lines comprising a third complementation group have a number of biochemical characteristics different from classical ML III and may represent a genetically distinct disorder.
Intronless human dihydrofolate reductase genes are derived from processed RNA molecules.
Authors: Chen M J , Shimada T , Moulton A D , Harrison M , Nienhuis A W
Published: May 2006

Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (lambda hDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR. The other two human DHFR genes (hDHFR-psi 1 and hDHRF-psi 2) lack introns. hDHFR-psi 2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-psi 1 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region of DNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-psi 1 and hDHFR-psi 2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in lambda hDHFR-psi 2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences.
Tumor promoter phorbol 12-myristate 13-acetate induces a clastogenic factor in human lymphocytes.
Authors: Emerit I , Cerutti P A
Published: May 2006

The mechanism of the clastogenic action--i.e., the ability to induce chromosomal aberrations--of the tumor promoter phorbol 12-myristate 13-acetate (PMA) was investigated. PMA at 10 and 100 ng/ml induced the formation of a low molecular weight (less than 10,000) clastogenic factor (CF) in phytohemagglutinin-stimulated human blood and lymphocyte cultures. Bovine erythrocyte Cu-Zn superoxide dismutase strongly inhibited PMA clastogenicity, both the formation of CF and the action of previously formed CF. The nonsteroidal anti-inflammatory agents indomethacin, imidazol, and 5,8,11,14-icosatetraynoic acid inhibited PMA clastogenicity and the clastogenic activity of previously formed CF. These results suggest that superoxide radicals and stimulation of the arachidonic acid cascade play a role in PMA-induced clastogenicity and the mechanism of action of the CF. The CF may relate the initial interaction of PMA with the cell membrane to the genome.
Isolated poliovirus capsid protein VP1 induces a neutralizing response in rats.
Authors: Chow M , Baltimore D
Published: May 2006

Antibodies were raised in rats against the poliovirus type 1 capsid proteins, VP1, VP2, and VP3. Antibodies directed against VP1 from type 1 poliovirus (Mahoney) neutralized type 1 but not type 2 poliovirus. Antibodies raised against VP2 and VP3 failed to neutralize type 1 virus. Thus, VP1 appears to be a neutralizing antigen for poliovirus and in its denatured form presents to the immune system its neutralizing determinants.
12-O-tetradecanoylphorbol 13-acetate-inducible proteins are synthesized at an increased rate in Bloom syndrome fibroblasts.
Authors: Mallick U , Rahmsdorf H J , Yamamoto N , Ponta H , Wegner R D , Herrlich P
Published: May 2006

A set of proteins, which in normal fibroblasts were barely, if at all, detectable, were synthesized at an increased rate in fibroblasts from patients with Bloom syndrome (BS). The same set of proteins was induced in normal human fibroblasts by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). In BS cells, TPA caused a further 2-fold increase in the rate of synthesis. Production of these proteins was inhibited by the addition of fluocinolone acetonide to the culture medium. One of the proteins (XHF1) present at high levels in BS fibroblasts and in TPA-treated cells was also induced by irradiation with ultraviolet light. This protein was secreted into the culture medium. Most other TPA-inducible proteins were cytoplasmic. Among other human mutants prone to chromosome aberrations we found one of three tested cases of Fanconi anemia and one case of ataxia-telangiectasia that showed increased spontaneous rates of synthesis of the TPA-inducible proteins. In these cases, however, the induction by TPA was like that seen in healthy fibroblasts.
Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence.
Authors: Perlman D , Halvorson H O , Cannon L E
Published: May 2006

Saccharomyces cerevisiae strain FH4C carries a single invertase structural gene, SUC2, but produces distinct invertase mRNAs and polypeptides for the secreted and cytoplasmic forms of the enzyme. The two major invertase cell-free translation products are polypeptides of 60,000 daltons (p60) and 62,000 daltons (p62) and correspond to the nonglycosylated cytoplasmic form of invertase and the precursor of glycosylated secreted invertase, respectively. This paper describes amino acid sequence and peptide map analyses of invertase polypeptides. The peptide maps demonstrate that p62, p60, and the in vivo secreted polypeptide have significant structural homology. Sequence analysis, however, revealed differences between p62 and p60 at their amino termini. p62 contains an amino-terminal signal sequence of 19 amino acid residues that is specifically cleaved during secretion in a cell-free system to generate the secreted 87,000-dalton invertase glycopeptide gp87. This signal sequence is not present in p60. p60 synthesis begins with a methionine which can be aligned with a methionine at residue 21 in p 62. During translation, the p60 initiator methionine is removed and the newly generated amino terminus is acetylated. Based on peptide map similarities, partial amino-terminal sequence data, and common genetic origin, it is suggested that p60 and p62 have identical amino acid sequences carboxy-terminal to the p60 initiator methionine (residue 21 of p62). The reciprocal correlations of signal sequence with secretion and absence of signal sequence with cytoplasmic localization provide proof of the signal hypothesis for secreted proteins. Two mechanisms are proposed for the derivation of p60 and p62 from a single structural gene: alternative promoter sites, and differential processing of a single primary transcript.
Cyclic AMP-mediated control of meiosis: effects of progesterone, cholera toxin, and membrane-active drugs in Xenopus laevis oocytes.
Authors: Schorderet-Slatkine S , Schorderet M , Baulieu E E
Published: May 2006

Progesterone depressed rapidly (50% at 1 min) and persistently cyclic AMP (cAMP) concentration that had been elevated by cholera toxin in Xenopus laevis oocytes. cAMP remained below 1 pmol per oocyte (mean basal level) for approximately 1 hr and thereafter rose to approximately 120% of control values, while germinal vesicle (nucleus) breakdown did not occur. In the absence of cholera toxin, progesterone treatment for 6 hr maintained cAMP concentration below the basal level (but not lower than 80%), and germinal vesicle breakdown occurred. Experiments in the presence of phosphodiesterase inhibitors suggested that progesterone modulates adenylate cyclase activity. The maturation promoting factor, which is formed after 3-5 hr of progesterone treatment and provokes germinal vesicle breakdown after its injection into untreated oocytes, also decreased cAMP concentration, an observation that may explain its "autoamplification." Nonsteroidal inducers of meiosis reinitiation (e.g., propranolol, methoxyverapamil, mersalyl) diminished the cholera toxin-mediated accumulation of cAMP, in contrast to compounds devoid of meiotic-inducing capacity and antagonist to progesterone action, such as gammexane (an inositol analogue) and 5'-deoxy-S-(2-methylpropyl)-5'-thioadenosine (a methylase inhibitor), that increased the nucleotide level. The fine control, suggested by the effects of small changes in cAMP levels, gives evidence of great sensitivity to a critical determinant governing meiotic cell division.
Exon/intron organization and complete nucleotide sequence of an HLA gene.
Authors: Malissen M , Malissen B , Jordan B R
Published: May 2006

We have isolated and determined the sequence of a genomic clone containing the gene for a human class I transplantation antigen. The gene contains seven exons. The first five exons code respectively for a signal peptide, for the first, second, and third extracellular domains of the protein molecule, and for the transmembrane region. The cytoplasmic segment is encoded by part of the fifth and the sixth and the seventh exons. The structure of the protein encoded by this unit is closely homologous with known class I transplantation antigens.
Human prostate specific and shared differentiation antigens defined by monoclonal antibodies.
Authors: Frankel A E , Rouse R V , Herzenberg L A
Published: May 2006

Splenic lymphocytes of BALB/c mice immunized with membrane-enriched fractions of human benign prostatic hyperplasia tissues were fused with the NS-1 light chain-secreting murine myeloma cell line. This generated hybridoma cultures that secreted immunoglobulins reactive in solid-phase radioimmunoassays with membrane preparations of prostatic tissues but not with membrane preparations of apparently normal human liver, spleen, thymus, or erythrocytes. After further screening of immunoglobulin reactivities and cloning of cultures, eight monoclonal antibodies were chosen that demonstrated reactivity with human prostate tissues. These monoclonal antibodies could be placed into at least three major groups--epithelium-specific, polyepithelial, and stroma-specific--on the basis of differential binding to the surfaces of various component cells in the prostate and other epithelia. Two antibodies defined unique protein antigens specific for prostate epithelia that were not crossreactive with prostatic acid phosphatase or the recently described "prostatic antigens." These antibodies also detected antigens on malignant prostate tissues as well as other malignant tissues. Four antibodies defined three unique polyepithelial protein antigens (two of the antibodies were different isotypes defining the same protein). Each of the polyepithelial antigens was expressed on a different spectrum of normal epithelial tissues. Two displayed brain tissue crossreactivity, one was present on pancreas, and one was present on platelets. The two antibodies that detected prostatic stromal protein antigens showed different spectra of reactivities. One antibody reacted with apparently all prostatic stromal cells as well as endothelial cells in the prostate and other organs. The other antibody apparently reacted with all prostatic stromal cells as well as myoepithelial and muscle cells in other organs.
Interactions of dicyclohexylcarbodiimide with myelin proteolipid.
Authors: Lin L F , Lees M B
Published: May 2006

Dicyclohexylcarbodiimide (DCCD) is known to bind preferentially to a proteolipid subunit of proton-translocating systems and thereby to inhibit proton transport. In the present study we show that, in an aqueous medium, DCCD binds to the bovine white matter proteolipid apoprotein, the major protein of central nervous system myelin. The binding is dependent on time, temperature, and concentration and is not inhibited by the hydrophilic carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. By contrast, when the incubation is carried out in chloroform/methanol no labeling by DCCD is demonstrable. In isolated rat myelin, DCCD binds specifically to the proteolipid and not to the myelin basic proteins. Liposomes reconstituted with the myelin proteolipid apoprotein transport protons, as assayed by quenching of the fluorescence of 9-aminoacridine. Preincubation of proteolipid-containing liposomes with DCCD results in an inhibition of transport. These studies have important implications for a possible ionophoric function of the myelin proteolipid and for the occurrence of transport processes within myelin.
Emetine resistance in chinese hamster ovary cells is associated with an altered ribosomal protein S14 mRNA.
Authors: Madjar J J , Nielsen-Smith K , Frahm M , Roufa D J
Published: May 2006

Chinese hamster ovary cell mutants resistant to the translational inhibitor emetine (Emt B mutants) express a single genetically altered 40S ribosomal subunit protein, S14. that appears to account for their drug resistance. To determine whether Emt B mutation affect the structural gene for the S14 protein, we isolated mRNAs from several wild-type and Emt B mutant clones. We translated the mRNAs in a cell-free system derived from wheat germ, and we discerned the biosynthesis of 59 ribosomal proteins, including S14. In every case, poly(A)+ mRNA programmed the cell-free system to synthesize an S14 protein electrophoretically identical to the S14 extracted from the ribosomes of the corresponding cell line. Messages from two Emt B mutants (Emr-2 and Emr-2-2) specified S14s that were electrophoretically distinct from the wild-type protein. Thus, Emt B mutations were expressed in mutant cell mRNAs, apparently reflecting mutagen-induced changes in S14 structural genes.
Isolation and partial characterization of a 5'-nucleotidase specific for orotidine-5'-monophosphate.
Authors: El Kouni M H , Cha S
Published: May 2006

A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This enzyme orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-beta-D-ribofuranosylbarbituric acid 5'-monophosphate (BMF), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate and beta-glycerophosphate, all of which--with the exception of the 2' or 3' monophosphates, ribose 5'-phosphate, and beta-glycerophosphate--are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SF reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase had a Mr 53,000, Km value of 1 mM for OMP, and Vmax value of 49 nmol/min . mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular "free" OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from aortic aciduria.
Spacer mutations in the lac ps promoter.
Authors: Stefano J E , Gralla J D
Published: May 2006

Mutations have been constructed that delete either one or two base pairs near position -19 in the lac ps promoter. Deletion of either of two adjacent base pairs increases the rates of open complex formation by nearly on order of magnitude. Two promoters that have different single-base deletions are indistinguishable by either their rates of open complex formation or stability of the open complexes once formed. However, simultaneous deletion of both base pairs produces a promoter that forms complexes at a rate similar to that of the unmodified DNA sequence. The maximal rate of open complex formation is achieved at a spacer length of 17 base pairs, the most frequently occurring spacer length among promoters. These results suggest that the spacing between the two strongly conserved regions of sequence homology is an important determinant of the rate of open complex formation. A model is suggested that proposes that three important promoter elements, the -10 region, the -35 region, and the spacer region, act simultaneously to facilitate open complex formation by RNA polymerase.
Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine.
Authors: Jones P A , Taylor S M , Mohandas T , Shapiro L J
Published: May 2006

Three cytidine analogs containing modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2'-deoxycytidine and pseudoisocytidine) induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP; pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X chromosome retained in a mouse-human somatic cell hybrid. Between 0.1% and 8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but two other analogs, 5-fluoro-2'-deoxycrytidine and 5.6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X chromosome present in the hybrid, was found to be late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2'-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because these analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.
Specific glycoprotein antigens on the surface of insect and mammalian stages of Trypanosoma cruzi.
Authors: Nogueira N , Unkeless J , Cohn Z
Published: May 2006

Two major surface antigens on Trypanosoma cruzi, the causative agent of Chagas disease, have been described [Nogueira, N., Chaplan, S., Tydings, J., Unkeless, J. & Cohn, Z. (1981) J. Exp. Med. 153, 629-639]. One, a Mr 75,000 glycoprotein (GP), is specific for the culture forms (insect-host stages) of the organisms--epimastigotes and metacyclic trypomastigotes. The other, a Mr 90,000 GP, was found in vertebrate-host stages of the organisms--bloodstream-form trypomastigotes. We now report that these two major surface antigens of T. cruzi seem to be unrelated proteins, as judged by tryptic and chymotryptic peptide analysis. Antibodies were raised in rabbits against epimastigote or trypomastigote proteins which had been immunoprecipitated with human antisera. These trypomastigote and epimastigote protein antisera reacted only with the homologous immunogen, as determined by immunoprecipitation of surface-labeled organisms and by immunofluorescence. The Mr 75,000 GP is detected only in cultured (insect stage) epimastigotes and metacyclic trypomastigotes. The Mr 90,000 GP is only present in bloodstream-form trypomastigotes, amastigotes, and trypomastigotes obtained from infected muscle cells in vitro. Therefore, the insect and vertebrate stages of this species display distinctive surface GPs that can be identified by surface labeling and immunoprecipitation techniques in six strains of T. cruzi (Y, CL, Peru, Colombiana, SF-12, and SF-21) isolated from widely different areas of South America.
Recombined flanks of the variable and joining segments of immunoglobulin genes.
Authors: Höchtl J , Müller C R , Zachau H G
Published: May 2006

The mechanism of generating immunoglobulin light chain genes by rearrangement of variable (V), joining (J), and constant (C) gene segments is still unknown. It has been discussed mostly in terms of excision and deletion of the DNA between the recombined V and J gene segments. However, the finding of DNA digests from the mouse myeloma T of a fragment (called f-T) that contains the 3' flank of a V kappa and the 5' flank of a J1 gene segment argued against a simple deletion mechanism [Steinmetz, M., Altenburger, W. & Zachau, H. G. (1980) Nucleic Acids Res. 8, 1709--1720]. The origin of fragment f-T has now been investigated by cloning and determining the sequence of the germ-line V gene segment that apparently participated in its formation. Moreover, analogous fragments containing flanking sequences were isolated from the myelomas MOPC 173 and 41 (f-173 and f-41) and studied by sequence analysis. The f fragments appear to be recombination products of V--J rearrangements reciprocal to rearranged kappa genes but, at least in the cases of f-T and f-173, not of the rearranged V genes present in the same tumor cell. This fact is best explained by a sister chromatid exchange mechanism of V--J recombination because, by this model, the rearranged V genes and the reciprocal flank recombination products would segregate into different cells during the following mitosis. The possibility is suggested that there exists in lymphocyte differentiation more than one mechanism of V--J recombination.
Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes.
Authors: Pike L J , Gallis B , Casnellie J E , Bornstein P , Krebs E G
Published: May 2006

A431 cell membranes phosphorylate a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gly) in which residues 2--12 correspond to the sequence of the reported site of tyrosine phosphorylation in pp60src. Epidermal growth factor stimulates the phosphorylation of this peptide 2-fold over basal levels in a dose-dependent fashion. Phosphorylation is linear for approximately 3 min at 30 degrees C and occurs on the tyrosine residue. Kinetic analysis of the phosphorylation reaction indicates that epidermal growth factor increases the average Vmax from 3.8 to 7.5 nmol/min per mg and slightly decreases the average Km from 0.53 mM to 0.28 mM. A number of other peptides analogous to this tridecapeptide are also phosphorylated by A431 membranes. The data suggest that peptides with sequences similar to the site of tyrosine phosphorylation in pp60src are preferred substrates for the kinase in these membranes. Thus, the epidermal growth factor-stimulated protein kinase has the potential to interact with and phosphorylate pp60src. However, the A431 membranes also phosphorylate a tyrosine-containing peptide of totally unrelated sequence, suggesting that the kinase possesses a broad specificity for peptide phosphorylation that may not reflect its specificity with protein substrates.
In vitro
binding of gibberellin A
4
to extracts of cucumber measured by using DEAE-cellulose filters
Authors: Keith B. , Brown S. , Srivastava L. M.
Published: May 2006

A rapid method for assaying [
3
H]gibberellin A
4
bound to a soluble protein from cucumber hypocotyls by using DEAE-cellulose filter discs is described. The binding is saturable, reversible with unlabeled gibberellin A
4
, and has a half-life of association under nonequilibrium conditions at 0-4°C of 6-7 min. By using this assay, the dissociation constant (
K
d
) was estimated to be 70 nM and the number of binding sites, 0.4 pmol mg
-1
of soluble protein or 0.69 pmol g
-1
(fresh weight) of hypocotyls. The speed and reliability of the assay make it invaluable for kinetic studies involving competitors. Thus, it has been possible to show that gibberellins that are biologically active in a cucumber bioassay compete for binding to the same protein and their calculated affinity constants bear a direct relationship to their known activities in the cucumber bioassay. Gibberellins that are inactive in this bioassay and other plant hormones, such as indoleacetic acid, abscisic acid, and kinetin, show a noncompetitive interaction.
Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.
Authors: Strome S , Wood W B
Published: May 2006

By using fluorescent antibody staining, we have followed cytoplasmic granules unique to germ-line cells throughout the life cycle of Caenorhabditis elegans. These elements, designated P granules, are segregated exclusively to germ-line precursor cells during early embryogenesis. Prior to mitosis at each of the early cleavages that produce a somatic and germ-line daughter cell, the granules become localized in the region of cytoplasm destined for the germ-line daughter. After the 16-cell stage, the granules appear to be associated with the nuclear envelope. P granules persist in the germ cells throughout the larval and adult stages. The P granules are similar in number, size, and distribution to germ-line-specific structures identified as "germinal plasm" by electron microscopy in C. elegans embryos.
Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.
Authors: Shortle D , Grisafi P , Benkovic S J , Botstein D
Published: May 2006

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.
Shared idiotypes among monoclonal antibodies specific for different immunodominant sugars of lipopolysaccharide of different Gram-negative bacteria.
Authors: Hiernaux J , Bona C A
Published: May 2006

Shared idiotypes (Ids) were identified on monoclonal antibodies specific for different immunodominant sugars in the lipopolysaccharide (LPS) of Gram-negative bacteria. The first group of monoclonal antibodies is specific for Escherichia coli 0113 LPS, whereas the second group is specific for Salmonella tranaroa LPS. The shared idiotopes borne by these two groups of monoclonal antibodies were identified with monoclonal anti-Id antibodies specific for E. coli LPS-binding monoclonal antibodies and with syngeneic anti-384Id antibodies specific for MOPC 384 and MOPC 870 myeloma proteins. Our data suggest that shared idiotopes are common for antibodies for an antigenic family.
Arachidonic acid metabolism in glutathione-deficient macrophages.
Authors: Rouzer C A , Scott W A , Griffith O W , Hamill A L , Cohn Z A
Published: May 2006

Mouse resident peritoneal macrophages were treated with the glutathione (GSH) synthesis inhibitor buthionine sulfoximine to deplete intracellular GSH. The arachidonic acid metabolites released by the GSH-depleted macrophages in response to a zymosan challenge were analyzed by HPLC. Buthionine sulfoximine treatment resulted in inhibition of both prostaglandin E2 and leukotriene C synthesis that was directly related to the degree of GSH depletion. Macrophages in which GSH levels were reduced to 3% of normal exhibited reductions to 4% and 1%, respectively, in PGE2 and LTC formation. The total quantity of cyclooxygenase metabolites secreted by GSH-deficient macrophages was identical to that of control cells as a result of increased synthesis of prostacyclin and, to a lesser extent, 12-L-hydroxy-5,8,10-heptadecatrienoic acid. Total lipoxygenase products were decreased, however; increased formation of hydroxyicosatetraenoic acids only partially compensated for the deficit in leukotriene C production. These findings extent our earlier observations on the inhibition of leukotriene C synthesis in GSH-depleted macrophages and confirm with intact cells the previously suggested role of GSH in prostaglandin E2 formation.
Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinomas from patients with epidermodysplasia verruciformis.
Authors: Ostrow R S , Bender M , Niimura M , Seki T , Kawashima M , Pass F , Faras A J
Published: May 2006

DNA extracted from squamous cell carcinomas from patients with the chronic wart disease syndrome, epidermodysplasia verruciformis, was analyzed for the presence of human papillomavirus (HPV)-specific DNA sequences by Southern blot hybridization analysis. Employing an HPV probe obtained by molecular cloning of viral DNA purified from benign warts from these patients, we have unequivocally identified HPV-specific nucleotide sequences in squamous cell carcinomas from these patients. Restriction endonuclease mapping indicated that the DNA present in the carcinomas was of the same type (type 5) as that found in the benign tumors from these patients and was present as unintegrated, free viral DNA. Moreover, we have demonstrated the presence of HPV-5 DNA in a subcutaneous metastatic tumor from one of these patients. This latter observation essentially eliminates the possibility that the HPV-5 DNA present in the malignant tumors in these patients resulted from cross-contamination from an adjacent benign warty lesion. In addition to wild-type HPV-5 DNA, both the primary and metastatic carcinomas analyzed also contained an HPV-5 DNA species lacking approximately 20% of the HPV-5 DNA genome. These subgenomic forms of HPV-5 DNA could not be detected in benign papillomas from these patients.
Lipoprotein lipase secretion by human monocytes and rabbit alveolar macrophages in culture.
Authors: Mahoney E M , Khoo J C , Steinberg D
Published: May 2006

Human peripheral blood monocytes and rabbit alveolar macrophages secreted lipoprotein lipase during culture. Within hours after plating, lipoprotein lipase had accumulated in the culture medium of monocytes, and the rate of accumulation increased with time in culture. The initial rate of secretion of lipoprotein lipase by alveolar macrophages was higher than in monocytes but decreased after 8--10 hr to values similar to those expressed by monocytes cultured for the same length of time. The enzyme was characterized as lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) on the basis of pH optimum (7.8--8.2 for monocytes, 8.1 for alveolar macrophages), dependence on apolipoprotein C-II for activity, inhibition by 0.3--0.5 M sodium chloride and protamine sulfate, and retention of a heparin-Sepharose gel. The expression of lipoprotein lipase secretion by human monocytes may have important implications with respect to the development of foam cells in the arterial wall during atherogenesis.
Perturbation and change in coral reef communities
Authors: Porter James W. , Battey James F. , Smith G. Jason
Published: May 2006

Ninety-six percent of surveyed shallow-water Dry Tortugas reef corals died during the severe winter of 1976-1977. Data from skeletal stains indicate that death occurred during the mid-January intrusion of 14°C water onto the reef. In deeper water, community parameters such as percent cover, species number, and relative abundance showed no significant change. However, an analysis of competitive interactions at the growing edges of adjacent colonies reveals a 70% reduction in space competition during this environmental disturbance. These results can explain high variability in the growth rate of Floridian reefs and demonstrate the importance of obtaining long-term spatial information to interpret successional dynamics of complex communities.
Nucleosome segregation at a defined mammalian chromosomal site.
Authors: Roufa D J , Marchionni M A
Published: May 2006

When animal cells replicate chromatin under conditions precluding new histone biosynthesis, half of the daughter DNAs are devoid of nucleosomes and are sensitive to staphylococcal nuclease. DNA sequences resistant to nuclease are associated with preexisting nucleosomes, which redistribute to progeny DNA duplexes during replication. We labeled newly replicated DNA sequences in a simian virus 40 (SV40)-transformed Chinese hamster cell clone with 5-bromodeoxyuridine (BrdUrd) in the presence and absence of a protein biosynthesis inhibitor, emetine. We resolved single-stranded BrdUrd- and dT-DNA sequences protected from nuclease digestion by nucleosomes and determined from which strands of the integrated viral DNA parental template (dT) and newly replicated progeny (BrdUrd) sequences were derived. Because we knew that the cell clone studied contained all of its integrated SV40 DNA at a single chromosomal site, we were able to determine that preexisting nucleosomes segregated to only one of the two daughter duplexes containing the integrated viral sequence. Additionally, in the presence of emetine, the integrated viral origin of replication, ORIsv, appeared not to function as a chromosomal replication origin, perhaps reflecting the drug's effect on synthesis of SV40 large tumor antigen.