bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.
Authors: Cohen A B , Gruenke L D , Craig J C , Geczy D
Published: May 2006

alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme.
Translational control by protein kinase in Artemia salina and wheat germ.
Authors: Sierra J M , de Haro C , Datta A , Ochoa S
Published: May 2006

The catalytic subunit of cyclic 3':5'-AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) inhibits translation in Artemia salina and wheat germ extracts. It acts, as in reticulocyte lysates [Datta, A., de Haro, C., Sierra, J. M. & Ochoa, S. (1977) Proc. Natl. Acad. Sci. USA 74, 1463-1467] by catalyzing the conversion of a proinhibitor to an inhibitor of polypeptide chain initiation. Addition of ATP and either cyclic AMP or catalytic subunit promotes the proinhibitor-inhibitor conversion in crude proinhibitor preparations from A. salina embryos. The effect of cyclic AMP is due to stimulation of cyclic AMP-dependent protein kinase, present in such preparations, and is inhibited by hemin. In similar preparations from wheat germ, addition of ATP and catalytic subunit promoted proinhibitor-inhibitor conversion, but addition of ATP and cyclic AMP has little or no effect. As assayed with histone as substrate, wheat germ preparations exhibit a protein kinase activity that is not stimulated by the addition of cyclic AMP or cyclic GMP. Our results suggest that a translational control system, similar to that existing in rabbit reticulocytes and other mammalian cells, is present in organisms evolutionarily far removed from mammals.
Choleragen activation of solubilized adenylate cyclase: Requirement for GTP and protein activator for demonstration of enzymatic activity
Authors: Moss Joel , Vaughan Martha
Published: May 2006

The requirements for choleragen activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] were investigated by using an enzyme preparation solubilized with Triton X-100 from an extensively washed brain particulate fraction and partially purified with DEAE-cellulose. Unlike the particulate enzyme, this preparation was not activated after incubation with choleragen plus dithiothreitol, ATP, and NAD. Addition of the purified protein activator of cyclic nucleotide phosphodiesterase and calcium to the partially purified enzyme increased basal activity somewhat, but choleragen activation was minimal. When cyclase was incubated with GTP plus the protein activator (and calcium), choleragen markedly increased the activity 3- to 6-fold. When GppNHp and protein activator were incubated with the cyclase prior to assay, activity was elevated but no effect of choleragen was observed. GTP and GppNHp had relatively small effects on cyclase activity in the absence of protein activator or if they were added directly to the assay. Boiled brain supernatant was consistently more effective than protein activator (plus calcium) and GTP, suggesting that other factors are required for maximal cyclase activity after choleragen treatment.

It appears that the cyclase system is dissociable into several components, all of which may be necessary for optimal regulation of activity. It is probable that one of these is the heat-stable calcium-dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase that we have found is required along with GTP for demonstration of choleragen activation of partially purified brain adenylate cyclase.
Computer analysis of nucleic acid regulatory sequences.
Authors: Korn L J , Queen C L , Wegman M N
Published: May 2006

We describe a computer program designed to facilitate the analysis of nucleic acid sequences. The program can search several nucleic acid sequences for oligonucleotides common to all of them. It can examine a DNA or RNA sequence for two kinds of homologous regions--repetitions and dyad symmetries. The homologies need not be perfect: mismatches and "looping out" of nucleotides are allowed. The program also finds (A+T)- and (G+C)-rich regions, locates restriction enzyme recognition sites, determines the distribution of di- and trinucleotides, and performs various other functions. We include two representative applications of the program. All published prokaryotic transcription termination sequences (June 1977) were found to share the following features: (i) a string of at least five T residues, (ii) the sequence CGGGC or a close analog immediately preceding the T cluster, (iii) a region of strong dyad symmetry preceding the Ts and including the CGGGC sequence. A sequence of 221 nucleotides consisting of the Escherichia coli trp promoter, operator, and leader was found to contain two strong dyad symmetries. These homologies both occur at known regulatory sites; no comparable homologies occur in regions without regulatory significance.
Induction and decay of human fibroblast interferon mRNA.
Authors: Cavalieri R L , Havell E A , Vilcek J , Pestka S
Published: May 2006

Polyadenylylated interferon mRNA, obtained from induced human fibroblasts, was quantitatively assayed by synthesis of biologically active human interferon in Xenopus laevis oocytes. The assay for interferon mRNA was used to distinguish between various hypotheses relating to interferon induction and biosynthesis. The data demonstrate that on induction with poly(I-poly(C) human fibroblasts accumulate interferon mRNA for 1-1.5 hr, after which time the mRNA is rapidly degraded with a half-life (t 1/2) of 18 min. Treatment of cells with cycloheximide prolongs the period of accumulation to 3 hr and decreases the rate of mRNA inactivation (t 1/2 = 49 min). Treatment with actinomycin D decreases the rate of inactivation still further (t 1/2 = 68 min). A comparison of cellular interferon synthesis with the relative amounts of interferon m RNA after simple induction or inductionin the presence of the inhibitors (superinduction) indicated a general correlation. Thus, on induction, the genes for interferon are activated to produce a transcript for a short time. The superinducing treatments prolong the period of accumulation and decrease the rate of degradation of this transcript.
Perturbation of chloroplast DNA amounts and chloroplast gene transmission in
Chlamydomonas reinhardtii
by 5-fluorodeoxyuridine
Authors: Wurtz Edwin A. , Boynton John E. , Gillham Nicholas W.
Published: May 2006

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in
Chlamydomonas
resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA appears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent.
Segregation at a locus determining an immunoglobulin genetic marker for the light chain variable region affects inheritance of expression of an idiotype
Authors: Laskin J. A. , Gray A. , Nisonoff A. , Klinman N. R. , Gottlieb P. D.
Published: May 2006

Previous investigations have demonstrated close genetic linkage between loci governing expression of strain-specific idiotypes and immunoglobulin heavy (H) chain allotype (i.e., the

C
H

locus). This linkage is presumed to reflect polymorphism of

V
H

genes (or of their expression) linked to the polymorphic

C
H

locus. That there was no apparent involvement of light (L) chain loci (thought to be unlinked to H chain) in inheritance of the idiotype-positive (Id
+
) phenotype was surprising, because the L chain is required for formation of each of the idiotypes studied at the chemical level. However, previous studies involving backcrosses of F
1
(Id
+
× Id
-
) mice to the Id
-
parental strain have never employed as the Id
-
parent one of several inbred strains shown by G. M. Edelman and P. D. Gottlieb [(1970)
Proc. Natl. Acad. Sci. USA
67, 1191-1199] to express a V
L
-region polymorphism. Among backcrosses performed in the present study, one involved the A/J strain as the Id
+
parent and the PL/J strain, one of the several strains with and L chain polymorphism, as the Id
-
parent. Whereas in three other backcrosses performed, idiotype expression segregated with H chain allotype, in the backcross to PL/J, all mice producing the characteristic A/J Id
+
phenotype were A/J allotype
+
, but not all A/J allotype
+
mice were Id
+
. Typing of backcross progeny for the Ly-3 thymocyte alloantigens, controlled by a locus closely linked to that governing the L chain polymorphism (called the
VK-1
locus), indicated that only the Ly-3 heterozygotes expressed the characteristic A/J Id
+
phenotype. Thus, all Id
+
backcross mice inherited both the H chain allotype and the
Ly-3
locus (and closely linked L chain-related locus) from the Id
+
A/J strain. This suggests that, when strains with L chain polymorphisms are included in genetic studies of idiotype expression, segregation of loci governing L chain expression may be found to contribute to inheritance of the Id
+
character. In addition, these studies may be taken as further evidence that the V
L
-region repertoire of the PL/J strain may differ considerably from that of most other inbred strains of mice.
On the enzymic defects in hereditary tyrosinemia.
Authors: Lindblad B , Lindstedt S , Steen G
Published: May 2006

The activity of the enzyme porphobilinogen synthase (EC 4.2.1.24) in erythrocytes from patients with hereditary tyrosinemia was less than 5% of that in a control group and the activity in liver tissue was less than 1% of the reported normal activity. Urine from patients with hereditary tyrosinemia contained an inhibitor that was isolated and identified as succinylacetone (4,6-dioxoheptanoic acid) by gas/liquid chromatography-mass spectrometry. Fresh urine samples contained succinylacetoacetate (3,5-dioxooctanedioic acid) as well as succinylacetone. The inhibition of porphobilinogen synthase explains the high excretion of 5-aminolevulinate observed in hereditary tyrosinemia. Succinylacetone and succinylacetoacetate presumably originate from maleylacetoacetate or fumarylacetoacetate, or both, and their accumulation indicates a block at the fumarylacetoacetase (EC 3.7.1.2) step in the degradation of tyrosine. We suggest that the severe liver and kidney damage in hereditary tyrosinemia may be due to the accumulation of these tyrosine metabolites and that the primary enzyme defect in hereditary tyrosinemia may be decreased activity of fumarylacetoacetase.
Comparison of Epstein-Barr viral DNAs in Burkitt lymphoma biopsy cells and in cells clonally transformed in vitro.
Authors: Sugden B
Published: May 2006

The nucleotide sequences of intracellular Epstein-Barr viral DNAs in tumor biopsy cells of clones independently transformed in vitro were generally compared by determing the electrophoretic mobilities of restriction endonuclease cleavage fragments of the viral DNA. To carry out this comparison, cleaved cell DNAs were electrophoresed in agarose gels and transferred to nitrocellulose paper, and the immobilized viral species were identified by hybridization with purified, viral DNA labeled in vitro. These studies lead to three findings: (i) The complexities of all of the intracellular viral DNAs are similar to one another and to that of purified virion DNA. (II) There are small differences in the cleavage patterns of some viral DNAs, but the differences of the cleavage patterns of the viral DNA resident in the tumor cells and in the cells transformed in vitro are not more pronounced than those found between the different clones of the cells transformed in vitro. (iii) All of the viral DNA species contain a repeated sequence. The first two conclusions indicate that the Epstein-Barr virus strain studied in the laboratory appears indistinguishable from that associated with Burkitt lymphoma.
Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle.
Authors: Froehner S C , Karlin A , Hall Z W
Published: May 2006

The acetylcholine receptor from denervated rat skeletal muscle was purified by affinity chromatography and, after reduction, was treated with the affinity alkylating agent 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The receptor specifically incorporated approximately 1 mol of alkylating agent per mol of 125I-labeled alpha-bungarotoxin bound. Analysis of the labeled receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that two subunits were labeled; their apparent molecular weights were 45,000 and 49,000. These results suggest that the affinity reagent labels a second site for acetylcholine binding in the muscle receptor that is not labeled in receptors from Electrophorus or Torpedo.
Structure determination of gaseous and amorphous substances by diffraction methods: Philosophical concepts and their implementation (A Review)
Authors: Karle Jerome
Published: May 2006

Structure determination by diffraction methods is reviewed in terms of the philosophical and mathematical aspects of the techniques used to investigate the atomic arrangements in gaseous and amorphous substances. Both theory and experiment must be adapted to each other's limitations. This is accomplished often only after much time and study have been devoted to the problems. Some areas of application are favored by an overdeterminacy in the data, which helps to simplify the mathematical aspects of the analysis. Other areas have relatively few data compared to the number of unknown parameters. In such cases, special analytical procedures that use, for example, chemical and structural information are indispensible. Applications cover a broad range of problems and afford fundamental information to many fields of science.
Nonintercalative binding of ethidium bromide to nucleic acids: crystal structure of an ethidium--tRNA molecular complex.
Authors: Liebman M , Rubin J , Sundaralingam M
Published: May 2006

X-ray diffraction studies at 4.5-A resolution on crystals of a complex of ethidium bromide and yeast phenylalanine tRNA reveal a nonintercalative model of binding of the ethidium within the tertiary structure. This is contrary to the expected interacalative binding to the double-helical regions.
Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.
Authors: Roberts J L , Herbert E
Published: May 2006

mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.
Response of endocytosis to altered fatty acyl composition of macrophage phospholipids.
Authors: Mahoney E M , Hamill A L , Scott W A , Cohn Z A
Published: May 2006

Mouse peritoneal macrophages incubated in serumless medium containing a 19:0 or trans-18:1 fatty acid complexed to bovine serum albumin incorporate the exogenous fatty acid supplement into cellular phospholipids. Within 8 hr, 25% of the total phospholipid fatty acids are derived from the supplement, with cell viability remaining greater than 95%. The incorporation of either of these supplements increases the saturated/unsaturated fatty acid ratio in the phospholipids 2-fold over that of cells cultured in serum and effects striking changes in endocytic activities. The levels of both fluid-phase pinocytosis and receptor-mediated phagocytosis are decreased at all temperatures examined between 15 degrees and 37 degrees. The increased degree of saturation of cell phospholipids correlates with decreased endocytic rates for both processes and with increased activation energies (Eact) for phagocytosis. The Eact values for phagocytosis, which range from 54 to 90 kcal/mol, depend on the supplementation conditions used. Although the levels of pinocytosis are depressed, the Eact values for pinocytosis (17--25 kcal/mol) are not strikingly affected by saturated fatty acid enrichment. These observations suggest that the degree of lipid fluidity of macrophage membranes influences both phagocytosis and pinocytosis in macrophages.
Isolation and characterization of the opioid peptides from rat pituitary: beta-endorphin.
Authors: Rubinstein M , Stein S , Udenfriend S
Published: May 2006

beta-Endorphin was isolated from 200 rat pituitaries by means of high-performance column chromatography, using sensitive fluorometric methods and a radioreceptor assay for opioid activity. The beta-endorphin was characterized as to molecular weight, amino acid composition, and mapping of tryptic peptides by a new microtechnique. It was found that rat beta-endorphin is identical to camel and sheep beta-endorphin. Furthermore, alpha-endorphin, Met-enkephalin, a nonpeptide morphine-like compound (MLC), and an additional unidentified opioid compound were detected in the extract of rat pituitary.
Identification of dynein as the outer arms of sea urchin sperm axonemes.
Authors: Ogawa K , Mohri T , Mohri H
Published: May 2006

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a tryptic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement.
Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells.
Authors: Schmidt-Ullrich R , Thompson W S , Lin P S , Wallach D F
Published: May 2006

Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.
Idiotypic regulation of the immune system by the induction of antibodies against anti-idiotypic antibodies.
Authors: Urbain J , Wikler M , Franssen J D , Collignon C
Published: May 2006

Anticarbohydrate antibodies (Ab1) were isolated from a rabbit hyperimmunized with Micrococcus lysodeikticus and injected into allotype-matched rabbits in order to obtain specific anti-iodiotypic antibodies (Ab2). Ab2 was isolated by means of a Sepharose column coupled to the anticarbohydrate antibodies and was injected into two allotype-matched rabbits. These latter rabbits produced specific anti-anti-idiotypic antibodies (Ab3) probably sharing idiotypic specificities with Ab1. However, these Ab3 did not react with the antigenic carbohydrate moiety of bacteria. The two rabbits that had produced Ab3 were then immunized with M. lysodeikticus and synthesized anticarbohydrate antibodies (Ab1') bearing idiotypic specificities similar to those of Ab1. The immune repertoire which is effectively expressed in one individual depends not only on the antigenic stimulation but also on the previous idiotypic history of the individual. These data support the concept that the immune system is a functional idiotypic network.
The nucleotide sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs.
Authors: Chang J C , Temple G F , Poon R , Neumann K H , Kan Y W
Published: May 2006

The complete sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs were determined by sequence analysis of full-length cDNAs. The single-stranded cDNAs were digested with the restriction endonuclease Hae III, and the two 3'-terminal fragments of 75 and 132 nucleotides, complementary to the 5' termini of the alpha- and beta-globin mRNAs, respectively, were isolated and sequenced. Including the initiation codon AUG, the untranslated 5' regions of human alpha- and beta-globin mRNAs contain 41 and 54 nucleotides, respectively, and exhibit striking homologies with the corresponding sequences in the rabbit. Human alpha- and beta-globin mRNAs have five bases in the region of the initiation codon that may form base pairs with the 3' terminus of 18S rRNA. Stable secondary structures with hairpin loops can be constructed in the untranslated 5' regions.
The 5'-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg.
Authors: Nomoto A , Kitamura N , Golini F , Wimmer E
Published: May 2006

The 5'-terminal, RNase T1-resistant oligonucleotide of poliovirus mRNA has been isolated. Its sequence is pU-U-A-A-A-A-C-A-Gp, which is identical to that of virion RNA except that the genome-linked protein VPg is absent [Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977) Nature 268, 208-213]. Because all newly synthesized viral RNAs are VPg-linked, we propose that VPg is cleaved from progeny RNA at the linkage between protein and nucleic acid prior to polyribosome formation. This may represent a new mode of processing of viral macromolecules. Virion RNA from which VPg has been cleaved proteolytically retains its specific infectivity, an observation suggesting that VPg is not involved in early steps (penetration and translation) of the infectious cycle initiated by RNA.
Cloning of the silk fibroin gene and its flanking sequences.
Authors: Ohshima Y , Suzuki Y
Published: May 2006

Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated. The starting material was DNA extracted from the posterior silk glands of Bombyx mori. Most clones were obtained from sheared DNA fragments linked by poly(dA)-poly(dT) joints to the plasmid pMB9. One of them includes the 5' end of the fibroin gene with a flanking sequence of 12 kilobases, and another includes the 3' end of the gene with a flanking sequence of about 1 kilobase. One clone was obtained by ligation, to pMB9, of a fragment generated by endodeoxy-ribonuclease EcoRI. This clone has a 21-kilobase insertion that probably includes the entire fibroin gene with flanking sequences at both ends. The cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.
Two distinct adenosine-sensitive sites on adenylate cyclase.
Authors: Londos C , Wolff J
Published: May 2006

The effects of adenosine and adenosine analogs on adenylate cyclases from several tissues have been examined. Two adenosine-reactive sites have been identified: (i) the "R" site, occupancy of which usually leads to activation of cyclase and which requires integrity of the ribose ring for activity, and (ii) the "P" site, which mediates inhibition and requires integrity of the purine ring for activity. Biphasic effects of adenosine are explained by the presence of both sites on a single adenylate cyclase. Comparison of these data with those in the literature indicates that adenosine-reactive "P" and "R" sites are present generally.
Mosaic mice with teratocarcinoma-derived mutant cells deficient in hypoxanthine phosphoribosyltransferase
Authors: Dewey Michael J. , Martin David W. , Martin Gail R. , Mintz Beatrice
Published: May 2006

Mutagenized stem cells of a cultured mouse teratocarcinoma cell line were selected for resistance to the purine base analog 6-thioguanine. Cells of a resistant clone were completely deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), the same X-linked lesion as occurs in human Lesch-Nyhan disease. After microinjection into blastocysts of another genetic strain, the previously malignant cells successfully participated in normal embryogenesis and tumor-free, viable mosaic mice were obtained. Cells of tumor lineage were identified by strain markers in virtually all tissues of some individuals. Mature function of those cells was evident from their tissue-specific products (e.g., melanins, liver proteins). These mutagenized teratocarcinoma cells are therefore developmentally totipotent. Retention of the severe HPRT deficiency in the differentiated state was documented in extracts of mosaic tissues by depressed specific activity of the enzyme, and also by presence of unlabeled clones in autoradiographs of explanted cells incubated in [
3
H]hypoxanthine. Some mosaic individuals had mutant-strain cells in only one or a few tissues. Such animals may provide unique opportunities to identify the tissue sources of particular aspects of the complex disease syndrome. The tissue distribution of HPRT-deficient cells suggests that selection against them is particularly strong in blood of the mosaic mice, as is already known to be the case in human heterozygotes. This phenotypic parallelism supports the expectation that afflicted F
1
male mice that might be obtained from mutant germ cells can serve as a model of the human disease.
DNA degradation in terminally differentiating lens fiber cells from chick embryos.
Authors: Appleby D W , Modak S P
Published: May 2006

During the terminal differentiation of lens fiber cells, nuclear DNA is known to accumulate free 3'-OH ends and is progressively lost from the nucleus. Toward the end of this process, nuclei undergo pycnosis and disappear. The size of the DNA in the epithelia and in early and late stages of fiber cell development was examined by electrophoresis on nondenaturing agarose/polyacrylamide gels. Low molecular weight DNA of discrete sizes appears only at the final stages of nuclear degeneration in central fiber cells and persists after the disappearance of the nuclei. These low molecular weight DNA fragments appear as multiples of a monomeric unit and are similar to the fragments produced by the digestion of epithelial cell nuclei by micrococcal nuclease. The data indicate that in lens fiber nuclei the double-strand breaks in vivo affect the chromatin during nuclear degeneration, and the data suggest that the DNA of these cells is organized into chromatin composed of discrete subunits.
Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity.
Authors: Jackson R C , Blobel G
Published: May 2006

The protease(s) responsible for removing the amino-terminal extension of nascent presecretory proteins (signal peptidase) has been extracted from rough microsomes of dog pancreas with the detergent sodium deoxycholate. Preprolactin and pre-growth-hormone, prepared by in vitro translation of bovine pituitary RNA in the wheat germ system, were used to assay signal peptidase in the extract. When added to the wheat germ system during translation, the extract reduced the size of preprolactin and pre-growth-hormone to that of prolactin and growth hormone, respectively. Post-translational addition of the extract also reduced the size of preprolactin and pre-growth-hormone to that of the authentic hormones. The prolactin produced by post-translational cleavage of radiolabeled preprolactin has been shown, by partial amino-terminal sequence analysis, to have the correct amino terminus. This post-translational assay has permitted the investigation of the subcellular localization of the enzyme. Sodium deoxycholate extracts of rough microsomes were active, whereas extracts of smooth microsomes were inactive. However, without detergent treatment, neither rough nor smooth microsomes were capable of cleaving preprolactin in the post-translational assay. From this we conclude that the signal peptidase activity is confined to the rough endoplasmic reticulum and is latent. Finally, we have detected two small peptides which we believe could be the signal peptides generated by the endoproteolytic cleavage of preprolactin and pre-growth-hormone by signal peptidase.
Controlling element-induced alterations in UDPglucose:flavonoid glucosyltransferase, the enzyme specified by the
bronze
locus in maize
Authors: Dooner Hugo K. , Nelson Oliver E.
Published: May 2006

The
bz
locus in maize, which directs synthesis of the enzyme UDPglucose:flavonoid glucosyltransferase (UFGT) in the endosperm, is one of many loci at which controlling element-induced phenotypic changes are known. To characterize the nature of such modifications, several
bz
mutants derived by transposition of the controlling element
Ds
to the
Bz
gene were analyzed. Three mutants were found to lack UFGT at all stages of endosperm development. Two others appeared to make an altered UFGT, and in one of these the enzyme showed an altered developmental profile.
Design of molecular control mechanisms and the demand for gene expression.
Authors: Savageau M A
Published: May 2006

Regulation by a repressor protein is the mechanism selected when, in the organism's natural environment, there is low demand for expression of the regulated structural genes. Regulation by an activator protein is selected when there is high demand for expression of the regulated structural genes. These general conclusions are useful in relating physiological function to underlying molecular determinants in a wide variety of systems that includes repressible biosynthetic pathways, inducible biosynthetic enzymes, inducible drug resistance, and prophage induction, as well as inducible catabolic pathways, for which a special case of this prediction previously was reported [Savageau, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 2453-2455].
Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.
Authors: Högberg-Raibaud A , Goldberg M E
Published: May 2006

Globular proteins often appear to consist of distinct compact "domains," and the assumption is frequently implicitly made that these domains correspond to intermediate structures in the folding process. If this assumption is correct, the polypeptide fragment that builds up a domain should be able to spontaneously fold into its native conformation even when isolated. In an attempt to isolate and study such a fragment, the beta2 subunit of tryptophan synthetase [tryptophan synthase, L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] has been subjected to controlled proteolysis. The resulting protein is shown to be a dimer, the protomer of which contains two nonoverlapping polypeptide chains of molecular weights 12,000 and 29,000. Though inactive, the nicked protein is shown to be in a conformation that closely resembles that of the original enzyme, since it still can form an enzyme-bound intermediate of the catalytic reactions. The fluorescence of this intermediate is used to characterize the binding sites for the cofactor (pyridoxal-P) and substrates, which are shown to exist on the nicked protein. The possibility is discussed of using the fragments isolated from the nicked protein to study individual steps of the enzymatic reaction, intracistronic complementation, and the folding process in the normal beta2 subunit.
Identification of a protein methyltransferase as the cheR gene product in the bacterial sensing system.
Authors: Springer W R , Koshland D E
Published: May 2006

Methylation of membrane-bound proteins with apparent molecular weights around 65,000 does not occur in mutants of the generally nonchemotactic cheR class of Salmonella typhimurium. This was shown to be due to the lack of a protein methyltransferase in these mutants by means of an in vitro assay using soluble proteins, membranes, and S-adenosylmethionine as the methyl donor. The methylase from the wild type was purified, characterized, and shown to be of molecular weight 38,000. It is specific for proteins in S. typhimurium and Escherichia coli membranes. The methylase is not required for tumbling but appears to be essential for maintaining the appropriate rate constants and levels of the regulator of the chemotactic response.
Identification of chlorophyll b in extracts of prokaryotic algae by fluorescence spectroscopy.
Authors: Thorne S W , Newcomb E H , Osmond C B
Published: May 2006

Solvent extracts of three different prokaryotic algae from three species of didemnid ascidians contained pigments identified, on the basis of their fluorescence excitation (E)and fluorescence emission (F)spectral maxima (measured in nm) at 77K, as chlorophyll a (E 449, F 678) and chlorophyll b (E 478, F 658). The release of algae on cutting or freezing Diplosoma virens was accompanied by a strong unidentified acid that converted these pigments to pheophytins. This unexpected finding provided further confirmation of the identity of the chlorophylls on the basis of the fluorescence spectra at 77K of pheophytin a (E 415, F 669) and pheophytin b (E 439, F 655). There was no evidence for the presence of the fluorescent bilin pigments found in other prokaryotic blue-green algae. Chlorophyll a/b ratios ranged from 2.6 to 12.0 in algae from different ascidians. The photosynthetic membranes were not organized into appressed thylakoids or grana in the algae from any of the three species of ascidians. The relationship between these observations and those in higher eukaryotic organisms is discussed.
Binding and internalization of thrombin by normal and transformed chick cells.
Authors: Zetter B R , Chen L B , Buchanan J M
Published: May 2006

Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The amount of cell-associated 125I-thrombin was found to be three times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 hr, whereas the trypsin-sensitive fraction is saturated after 1-4 hr. Autoradiography of thin sections of 125I-thrombin-treated cells observed by electron microscopy reveals that after 10 hr incubation greater than 70% of the label is localized in the cytoplasm of both normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in two large fragments of 22,000 and 19,500 daltons.
Secretion of alpha-ecdysone by crab Y-organs in vitro.
Authors: Chang E S , O'Connor J D
Published: May 2006

The cultured molting glands (Y-organs) from two species of crabs secrete a product whose behavior in several chromatographic systems and whose mass spectrum are identical with those of authentic alpha-ecdysone. It appears that alpha-ecdysone is the only molting hormone secreted by the Y-organ, and that the Y-organ is the sole source of alpha-ecdysone in these animals.
Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40.
Authors: Schmidt-Ullrich R , Thompson W S , Wallach D F
Published: May 2006

Highly purified plasma membranes from hamster lymphocytes transformed by simian virus 40 (GD 248) were compared with the membranes of normal cells by crossed immune electrophoresis, crossed-line immune electrophoresis, and bidimensional isoelectric focusing-immune electrophoresis. Antiserum raised by inoculation of guinea pigs with GD 248 membranes was used as serologic reagent, either directly or after absorption with membranes from normal cells. Bidimensional immune electrophoresis reveals the presence in the plasma membranes of GD 248 cells of at least three antigens not detectable in the membranes from the normal cell population. At least two of these are also present in the mitochondrial membranes of GD 248 cells, but none could be detected in membranes of embryonic fibroblasts. Bidimensional isoelectric focusing-immune electrophoresis indicates that the distinctive antigens of the GD 248 membranes are glycoproteins.
Deletions affecting the transposition of an antibiotic resistance gene.
Authors: Heffron F , Bedinger P , Champoux J J , Falkow S
Published: May 2006

The structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 X 10(6) dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 X 10(6) dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 X 10(6) dalton recombinant plasmid by a combination of nuclease treatments in vitro. From this set of deletions a subset was isolated that contained deletions affecting the transposition of TnA. The deletions were mapped by digestion with restriction nucleases and electron microscopic analysis of DNA hetero-duplexes and were found to include one of the inverted repeated sequences or lie in the central portion of TnA. Complementation experiments were attempted between these plasmids and another compatible plasmid carrying a deletion in TnA that abolished its ampicillin resistance. The results of the deletion data indicate that approximately 2 X 10(6) daltons of TnA is required for transposition; the complementation experiments suggest that the terminal inverted repetition and the central region of TnA play different essential roles in TnA transposition.
Inhibition of proliferative and plaque-forming cell responses by human bone-marrow-derived lymphocytes from peripheral blood by antisera to the p23, 30 antigen.
Authors: Friedman S M , Breard J M , Humphreys R E , Strominger J L , Schlossman S F , Chess L
Published: May 2006

A recently described technique for the polyclonal induction of plaque-forming cells from human bone-marrow derived (B) lymphocytes of peripheral blood has been used to assess the role of a human Ia-like antigen (p23,30) in differentiation of human B cells. In particular, the effects of antisera to p23,30 on the plaque-forming cells and proliferative responses of human B cells stimulated by pokeweed mitogen or soluble products of activated human thymus-derived lymphocytes (T cells) have been examined. Antisera to p23,30 eliminated the development of plaque-forming cells induced by both T cell products and pokeweed mitogen. While these antisera also abrogated B cell proliferation induced by T cell supernatants, the proliferative response generated by pokeweed mitogen was only partially reduced. It was also determined that while the p23,30 antigen continues to be expressed on fully differentiated plaque-forming cells, antisera to this determinant exert inhibitory effects on B cell differentiation only when present during the early stages of B cell cultures. These results lend further support to the analogy between p23,30 and murine Ia antigens. Moreover, they demonstrate a major role for this antigen in the early events involved in human B cell differentiation into antibody-forming cells.
Biological function of gramicidin: studies on gramicidin-negative mutants.
Authors: Mukherjee P K , Paulus H
Published: May 2006

By the use of a rapid radioautographic screening procedure, two mutants of Bacillus brevis ATCC 8185 that have lost the ability to produce gramicidin have been isolated. These mutants produced normal levels of tyrocidine and sporulated at the same frequency as the parent strain. Their spores, however, were more heat-sensitive and had a reduced dipicolinic acid content. Gramicidin-producing revertants occurred at a relatively high frequency among the survivors of prolonged heat treatment and had also regained the ability to produce heat-resistant spores. A normal sport phenotype could also be restored by the addition of gramicidin to cultures of the mutant strain at the end of the exponential growth. On the other hand, the addition of dipicolinic acid could not cure the spore defect. These results provide strong evidence that the inability to produce gramicidin is directly responsible for the observed spore defects. Indeed, they unambiguously demonstrate a function of a peptide antibiotic in bacterial sporulation. The possibility that this function consists of the regulation of transcription during the transition from growth to sporulation is discussed.
Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood.
Authors: Clarke B J , Housman D
Published: May 2006

We have found that the peripheral blood of normal humans contains a significant number of committed erythroid stem cells of high proliferative capacity. These erythroid stem cells closely resemble the murine erythroid burst-forming unit (BFU-E) with respect to proliferative capacity, colony morphology, and erythropoietin requirement. BFU-E were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque density gradient centrifugation and cultured in vitro using the plasma culture technique. Macroscopic erythroid colonies of between 100 and 1000 cells were observed after 10-14 days of culture in the presence of either sheep or human erythropoietin at 0.5-4 units/ml. Individual colonies contained between 3 and 20 subcolonies and reached a maximum mean size of approximately 500 cells. Colony number was linearly related to the cell input, suggesting that a single cellular entity was precursor to each colony. The frequency of cells capable of giving rise to an erythroid colony was at least 100 per ml of blood in a number of individuals tested. The ability to assay significant numbers of erythroid precursor cells of high proliferative capacity from normal peripheral blood should facilitate the study of both normal erythropoiesis and of disease states affecting erythropoiesis in which marrow samples are not available on a routine basis.
Eukaryote ribosomes possess a binding site for concanavalin A.
Authors: Howard G A , Schnebli H P
Published: May 2006

Ribosomes prepared from chicken liver or rabbit reticulocytes bound concanavalin A in a molar ratio of approximately 1:1. This binding is to the large subunit of the eukaryote ribosomes with a dissociation constant of 5 X 10(-7) M (0 degrees). The binding of concanavalin A to Escherichia coli ribosomes was much less. Binding to the RNA or to possible membrane contaminants was ruled out in control experiments. Chicken liver ribosomes were labeled in vivo with 3H-labeled amino acids, purified, and dissociated in sodium dodecyl sulfate. Affinity chromatography of this preparation made it possible to isolate the small proportion of the ribosomal proteins (about 1.5%) containing the concanavalin A binding site. This protein moved as a single band during electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and showed an apparent molecular weight of 31,000.
Purification of a carbonic anhydrase from the inner ear of the guinea pig.
Authors: Drescher D G
Published: May 2006

A soluble carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified to homogeneity from the membranous lateral wall (stria vascularis, spiral ligament, spiral prominence, and outer sulcus) of the guinea-pig inner ear. About 1% of the protein of the membranous lateral wall is carbonic anhydrase. The specific activity of the enzyme in homogenates of the lateral wall is 1.6-1.8 times that of whole blood; for homogenates of the components, stria vascularis and the fraction containing the spiral ligament, the specific activities are 0.9 and 2.0 times the specific activity of whole blood, respectively. No other cochlear fraction examined contains appreciable carbonic anhydrase. The purified enzyme has a molecular weight of about 30,000, a specific activity 60--80% that of carbonic anhydrase C from blood, and an electrophoretic mobility similar to that of the blood enzyme. Cochlear carbonic anhydrase is half-maximally inhibited by 4 X 10(-9) M acetazolamide, is completely inhibited above 10(-5)M acetazolamide, and forms a fluorescent complex with 5-dimethylaminonaphthalene-1-sulfonamide, by which it can be distinguished on polyacrylamide gels. This report describes both another isolation of a carbonic anhydrase from a source other than blood and the isolation of an inner-ear enzyme.