bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal-binding and detoxification effect of synthetic oligopeptides containing three cysteinyl residues.
Authors: Yoshida A , Kaplan B E , Kimura M
Published: May 2006

Metallothionein is a naturally occurring metal-binding protein with high cysteine content. Oligopeptides containing three cysteinyl residues and having amino acid sequences analogous to portions of this protein were synthesized by the solid-phase method. Strong affinity of the synthetic peptides to Cd2+ and Zn2+ was observed, and the dissociation constants of the peptide-metal complexes were 2-4 orders of magnitude lower than those of cysteine-metal and dithioerythritol-metal complexes. Effectiveness of detoxification of the peptides against Cd toxicity was demonstrated by the higher survival rates of mice treated with the peptides and by the neutralization of Cd toxicity by the peptides in tissue cultures.
Appearance of catecholamine-synthesizing enzymes during development of rat sympathetic nervous system: Possible role of tissue environment
Authors: Teitelman Gladys , Baker Harriet , Joh Tong H. , Reis Donald J.
Published: May 2006

We sought to determine, in rat embryo, when and at what site in their migration cells derived from the neural crest differentiate into sympathetic neuroblasts. This has been accomplished by immunocytochemical detection, within the cells, of the enzymes catalyzing catecholamine biosynthesis—tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] dopamine-β-hydroxylase [DBH; 3,4-dihydroxyphenylethylamine,ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1)]—and, as a marker of prospective adrenal medullary cells, the enzyme phenylethanolamine
N
-methyltransferase (PNMT;
S
-adenosyl-L-methionine:phenylethanolamine
N
-methyltransferase, EC 2.1.1.28). TH and DBH, not detected in the neural crest, appear almost simultaneously in cells of the thoracic sympathetic ganglia in 11-day-old embryos, and in abdominal and lumbar ganglia 1-2 days later, thereby exhibiting a characteristic rostral-caudal gradient of differentiation. Cells stained for TH and DBH are seen in the gut wall from day 11 to day 14, but not thereafter. Cells stained for TH and DBH appear in the adrenal anlage at day 15. However, PNMT is not detected in the adrenal until day 17 of development, and is present only in the sympathoblasts in contact with the adrenal cortex. Treatment of pregnant rats with dexamethasone failed to accelerate the appearance of PNMT in the embryo or to initiate its expression in cells of other sympathetic organs. We conclude that neural crest cells express a noradrenergic phenotype only after leaving the neural crest and that these cells are labile with respect to their neurotransmitter and are capable of transformation in response to environmental stimuli.
Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.
Authors: Blanchard S G , Raftery M A
Published: May 2006

Procaine amide azide, a derivative of the local anesthetic procaine amide, was prepared and its interactions with acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax were studied. Procaine amide azide was radiolabeled by a method that may be of general use for the preparation of other radioactive tertiary amines. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of receptor-rich membrane preparations, a simple pattern of incorporated radioactivity was seen after electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. Only two major labeled bands were seen, corresponding to apparent Mr of about 43,000 and 90,000. When the length of the gels was increased, the labeled band of lower Mr was resolved into two labeled proteins, one major and one minor, with apparent Mr of 43,000 and 40,000, respectively. The radioactivity incorporated into the protein of Mr 40,000 could be attributed to interaction of [3H]procaine amide azide with cholinergic ligand-binding sites, whereas labeling of the polypeptide of Mr 43,000 appears to represent interaction of the photolabile derivative with another class of sites. The labeling component of 90,000 Mr could be removed by preparation of membrane fragments in iodoacetamide-containing buffer and therefore appeared unrelated to the acetylcholine receptor.
Effects of ethanol on alanine metabolism in perfused mouse liver studied by 13C NMR.
Authors: Cohen S M , Shulman R G , McLaughlin A C
Published: May 2006

Time courses of 13C labeling from alanine and ethanol in perfused mouse livers have been followed by NMR. The enrichment at specific carbons of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, beta-hydroxybutyrate, and lactate has been measured. The specific labeling of glutamate in the presence of labeled alanine and labeled or unlabeled ethanol shows that, under these conditions, alanine enters the tricarboxylic acid cycle almost exclusively through pyruvate carboxylation, whereas ethanol is the exclusive source of acetyl-CoA. In the absence of ethanol, the alanine label flows through both paths. By comparing the scrambling of 13C between C3 and C2 of glutamate it is possible to estimate the mitochondrial fumarase activity; the C6-to-C5 ratios in glucose give the additional scrambling by cytosolic fumarase activity. In addition, the C6-to-C1 and C5-to-C2 ratios in glucose show that there is about 15% flux through the pentose cycle. Finally, the C4-to-C2 ratios in glutamine and glutamate are unequal at any time (the glutamine labels reflect the label distribution in glutamate measured 1 hr earlier), providing a method for studying flow through glutamine synthetase in situ.
Interferon action: induction of specific proteins in mouse and human cells by homologous interferons.
Authors: Gupta S L , Rubin B Y , Holmes S L
Published: May 2006

Treatment of mouse (Ehrlich ascites tumor and L929) and human (FS4, GM258, etc.) cells with homologous interferons results in the induction of several proteins. Extracts obtained from cells labeled with [35S]methionine in the absence or presence of interferon were fractionated on poly(I) . poly(C)-agarose columns. The proteins retained on the columns revealed, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, three protein bands in mouse cells (Mr 120,000; 80,000; and 67,000) and two in human cells (Mr 120,000 and 80,000) which were detected in the extracts of interferon-treated but not of untreated cells. These proteins were retained on double-stranded RNA [poly(I) . poly(C)-agarose] columns but very poorly, if at all, on single-stranded RNA [poly(I)- or poly(C)-agarose] columns, suggesting that they have an affinity for double-stranded RNA. In addition, interferon treatment of human fibroblasts greatly increased the labeling of three other protein bands (Mr 88,000; 67,000; and 56,000) which were detected in whole extracts but were not appreciably retained on poly(I) . poly(C)-agarose columns. The appearance of the induced proteins was blocked by actinomycin D if added together with interferon, indicating that transcription of certain genetic information is required. The possible correlation between the induced proteins described here and the elevated levels of certain enzymes in interferon-treated cells (a protein kinase and 2'-5'-oligoadenylate synthetase) is at present unclear.
Studies of binding of prekallikrein and Factor XI to high molecular weight kininogen and its light chain.
Authors: Thompson R E , Mandle R , Kaplan A P
Published: May 2006

Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW)-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 x 10(7) M-1 and 4.2 x 10(8) M-1, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMW-kininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity.
Tryptic peptide analysis and NH
2
-terminal amino acid sequences of polyhedrins of two baculoviruses from
Orgyia pseudotsugata
Authors: Rohrmann G. F. , Bailey T. J. , Brimhall B. , Becker R. R. , Beaudreau G. S.
Published: May 2006

Comparative analysis of the tryptic peptides and terminal amino acid sequence was made on polyhedrins from two genetically different baculoviruses that are naturally pathogenic for the same insect host. Comparison of the tryptic peptides of the nucleopolyhedrosis bundle virus and nucleopolyhedrosis single-rod virus of
Orgyia pseudotsugata
by means of cation-exchange resins indicated that the proteins have a closely related amino acid sequence. The NH
2
-terminal amino acid sequence of polyhedrins from the two viruses differed in only 4 out of 34 amino acids. The nucleopolyhedrosis bundle virus and the nucleopolyhedrosis single-rod virus also differed in 4 and 5 out of 34 terminal amino acids, respectively, from the sequence reported for polyhedrin of a baculovirus of
Bombyx mori
[Serebryani, S. B., Levitina, T. L., Kautsman, M. L., Radavski, Y. L., Gusak, N. M., Ovander, M. N., Sucharenko, N. V. & Kozlov, E. A. (1977)
J. Invertebr. Pathol.
30, 442-443]. In addition, the nucleopolyhedrosis single-rod virus had two amino acids (Met-Tyr) on the NH
2
terminus that were not present on the terminus of nucleopolyhedrosis bundle virus or
B. mori
baculovirus polyhedrin. Approximately half (six) of the total tyrosine residues are clustered in the terminal 20 amino acids of the polyhedrins. Secondary structures predicted from the primary sequence suggest that the tyrosines are clustered in two areas. This nonrandom distribution and the pK
a
of about 10 for tyrosine may be related to the alkali solubility of the polyhedrin.
Regulation of activity of chromatin receptors for thyroid hormone: possible involvement of histone-like proteins.
Authors: Eberhardt N L , Ring J C , Johnson L K , Latham K R , Apriletti J W , Kitsis R N , Baxter J D
Published: May 2006

Thyroid hormone receptors lose their capability for high-affinity binding of the biologically active triiodothyronine after solubilization and separation from other chromatin proteins. The high-affinity triiodothyronine-binding capacity can be reconstituted by addition of a histone-containing extract of chromatin of purified core histones (H2A, H2B, H3, and H4); a number of other acidic or basic proteins tested were ineffective. The data support a model of the receptor in which a "core" receptor subunit that contains a thyroid hormone-binding site interacts with a regulatory subunit, which is possibly a histone or histone-like species. This interaction with the "core" subunit enables the resulting "holo" receptor to bind biologically active hormones. These data also suggest that histones or related proteins can modulate the activity of nonhistone chromosomal proteins that are involved in regulating the expression of specific genes.
Interactions between DNA-bound repressors govern regulation by the λ phage repressor
Authors: Johnson Alexander D. , Meyer Barbara J. , Ptashne Mark
Published: May 2006

The λ phage repressor binds cooperatively to the three sites in the right operator (
O
R
) according to the following pattern. If the DNA is wild type,
O
R
1 and
O
R
2 are filled coordinately because of interactions between repressor dimers bound to these two sites. Site
O
R
3 is filled only at higher repressor concentrations. In contrast, if
O
R
1 is mutant,
O
R
2 and
O
R
3 are filled coordinately because of interactions between repressors bound to these sites. In this case, the affinity of
O
R
3 is increased and that of
O
R
2 is decreased relative to the wild type. We infer that a repressor dimer bound to the middle site
O
R
2 can interact either with another repressor dimer bound to
O
R
1 (wild-type case) or, alternatively, with one bound to
O
R
3 (mutant
O
R
1 case). We argue that these repressor interactions are mediated by protein-protein contacts between adjacent repressor dimers, because the isolated amino-terminal domains of repressor bind to the operator sites noncooperatively. The
cro
protein of phage λ, a second regulatory protein, which recognizes the same three sites in
O
R
as does repressor, binds non-cooperatively. Experiments performed
in vivo
show that regulation of gene expression by repressor can be influenced critically by cooperative interactions. We demonstrate that the effect of repressor in a lysogen on the activity of the promoter
P
RM
can be changed from activation to repression by deletion of
O
R
1. We explain this effect in terms of the alternative cooperative interactions described above.
Mechanism of tumor promoter inhibition of cellular binding of epidermal growth factor
Authors: Lee Lih-Syng , Weinstein I. Bernard
Published: May 2006

In previous studies we demonstrated that the tumor-promoting agent 12-
O
-tetradecanoyl phorbol 13-acetate (TPA) and related macrocyclic diterpenes are potent inhibitors of the binding of epidermal growth factor (EGF) to its cell surface receptors in HeLa cells. The present study explores the specificity and mechanism of this effect. We have found that the same effect is observed in various cell types derived from mice, rats, or humans. In HeLa cells TPA inhibits the initial binding of EGF and also accelerates the loss of previously bound EGF from cells. The released EGF is recovered largely intact in the medium, indicating that TPA does not induce increased proteolysis or increased cellular internalization and degradation of EGF. The TPA effect on EGF receptors is mediated by a highly temperature-dependent process because TPA inhibition of EGF binding, and TPA-induced release of prebound EGF, are much greater at 37°C or 22°C than at 4°C. A curious feature is that when cells are grown in TPA for one or more days they escape or become refractory to TPA inhibition of EGF binding. Taken together, these results suggest that TPA inhibits EGF binding not by binding directly to the “active site” of the EGF receptor but by indirectly altering the conformation or inducing the clustering of EGF receptors. These and other membrane effects of this tumor promoter suggest that it is a valuable probe for elucidating complex aspects of membrane structure and function.
Adrenocorticotropin (ACTH) induces phosphorylation of a cytoplasmic protein in intact isolated adrenocortical cells.
Authors: Podesta E J , Milani A , Steffen H , Neher R
Published: May 2006

In 32P incorporation experiments with intact adrenocortical cells, adrenocorticotropin (ACTH) or adenosine 3',5'-cyclic monophosphate (cAMP) induced a rapid and transient increase of approximately 300-500% in the phosphorylation of a 32P-containing cytoplasmic protein of about 150,000 daltons (APS150). Half-maximal stimulation of APS150 phosphorylation was observed with about 3 pM ACTH. Receptor-bound cAMP, corticosterone production, and the appearance of phosphorylated APS150 increased in parallel with respect to both time and ACTH concentration. All three responses were dependent on extracellular calcium. Inhibition of protein synthesis with cycloheximide suggested a half-life of APS150 of about 10 min. The time course of 32P incorporation into ACTH-induced APS150 in the absence and presence of nonradioactive phosphate shows that the phosphorylation of APS150 is under simultaneous control of cAMP-dependent protein kinase and of phosphoatase activity. Thus a rapid ACTH-dependent and cAMP-dependent protein phosphorylation in intact adrenocortical cells within steroidogenic ACTH concentrations has now been demonstrated.
Tumor cell variants obtained by mutagenesis of a Lewis lung carcinoma cell line: immune rejection by syngeneic mice.
Authors: Van Pel A , Georlette M , Boon T
Published: May 2006

It has been reported that, by mutagenesis of a malignant mouse teratocarcinoma cell line, it is possible to obtain cell variants that are incapable of forming progressive tumors in syngeneic mice. These variants, which were called "tum-," are eliminated from the host by an immune rejection process. We report here that similar variant cell clones can be obtained at high frequency from a Lewis lung carcinoma cell line treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Syngeneic C57BL/6 mice reject these tum- clones and acquire a strong radioresistant immune protection against the immunizing clone. When the challenging tum- clone differs from the immunizing clone, a weaker radioresistant immune protection can be demonstrated with some, but not all, combinations. All the tum- clones induce a significant protection against the original Lewis lung malignant cells. These results imply that each Lewis lung tum- variant carries on its surface a singular antigen in addition to one or more weak antigens already present on the original tumor cell line. This antigenic pattern is similar to that found on teratocarcinoma tum- variants. Our results suggest that the procedure of using a mutagen in order to generate tum- variants carrying new transplantation antigens may be generally applicable to cancer cells.
Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly.
Authors: Karr T L , Podrasky A E , Purich D L
Published: May 2006

Critical concentrations for formation of microtubules from subunits with GTP and its beta, gamma-imido and beta, gamma-methylene analogs are similar when adequate time is given for equilibration. Dilution of microtubules into GTP and GDP yielded values of 0.1 and 0.19 mg/ml for the critical concentration, results similar to those reported by Carlier and Pantaloni [Carlier, M. & Pantaloni, D. (1978) Biochemistry 17, 1908-1915]. GDP is capable of supporting elongation of preformed microtubules, but it efficiently poisons the nucleation events. Reported experiments also demonstrate that the critical tubulin concentration of the tubulin-GDP complex can be accurately measured in both the assembly and disassembly directions. Evidence is presented that GTP is involved in early nucleation events but that microtubules are stabilized in the presence of either GTP or GDP. These results are discussed in terms of a condensation-equilibrium model in which tubulin subunits equilibrate rapidly with microtubule ends, and their affinity for the ends is governed by the nucleotide ligand at the exchangeable site.
Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus
Authors: Lerner Michael Rush , Steitz Joan Argetsinger
Published: May 2006

Patients with systemic lupus erythematosus often possess antibodies against two nuclear antigens called Sm and RNP (ribonucleoprotein). We have established the molecular identity of these antigens by analyzing immune precipitates of nuclear extracts from mouse Ehrlich ascites cells labeled with
32
P and
35
S. Anti-Sm serum selectively precipitates six small nuclear RNA molecules (snRNAs); anti-RNP serum reacts with only two of these; and a third serum, characterized as mostly anti-RNP, precipitates a subset of three snRNA bands. Three of the six RNAs are identified by fingerprint analysis as the previously characterized and highly abundant nucleoplasmic snRNA species U1a (171 nucleotides), U1b, and U2 (196 nucleotides). The other three RNAs (U4, U5, and U6) likewise are uridine rich and contain modified nucleotides, but they are smaller, with lengths of about 145, 120, and 95 residues, respectively. Each of the six snRNAs is complexed with and apparently antigenic by virtue of association with specific proteins. All three sera precipitate an identical complement of seven different polypeptides ranging in molecular weight from 12,000 to 35,000; these proteins are abundant in nuclear extracts, but are neither histones nor the major polypeptides comprising the 30S heterogeneous nuclear RNP particles of mammalian nuclei. Our data argue that each of the six snRNAs exists in a separate small nuclear ribonucleoprotein (snRNP) complex with a total molecular weight of about 175,000. We find that human antisera also precipitate snRNAs from a wide range of vertebrate species and from arthropods. We discuss the antigenic snRNPs in relation to the published literature on snRNAs and nuclear RNPs and consider possible functions of snRNPs in nuclear processes.
Myxobacterial hemagglutinin: A development-specific lectin of
Myxococcus xanthus
Authors: Cumsky Michael , Zusman David R.
Published: May 2006

Fruiting body formation in the bacterium
Myxococcus xanthus
consists of a temporal sequence of cellular aggregation and sporulation. During the period of cellular aggregation, a major new development-specific protein that has lectin-like activity is synthesized. This protein, called myxobacterial hemagglutinin (MBHA), was able to agglutinate sheep or guinea pig erythrocytes but not horse, ox, chicken, or human erythrocytes. MBHA was undetectable in extracts of vegetative cells, cells starved in liquid buffer, or in glycerol-induced cells. However, cells starved on a fruiting medium produced large amounts of MBHA (about 5% of protein synthesis), starting at about 6-8 hr of development. The protein accumulated in the soluble fraction of cells, reaching a peak of 1-2% of total protein at about the time when aggregation was completed. At later times the amount of MBHA present in the soluble fraction declined although synthesis continued. The hemagglutinating activity of MBHA could not be inhibited with simple sugars or aminosugars but could be inhibited with fetuin, a fetal calf serum glycoprotein. The
O
-glycosidically linked trisaccharide glycopeptide of fetuin was shown to be inhibitory by itself. The penultimate galactose of this glycopeptide was directly implicated in the inhibitory activity, because the inhibition by asialofetuin was reduced to 1/60th by periodate oxidation and to 1/15th after β-galactosidase treatment. MBHA is an abundant biochemical marker of development in
M. xanthus
. The fact that it is a lectin suggests that it may play a role in cell—cell recognition or agglutination.
Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis.
Authors: Mallick U , Herrlich P
Published: May 2006

Cyclic AMP is the effector molecule for both positive and negative control of synthesis of several Escherichia coli proteins. Among the latter is the major outer membrane protein III. The control mechanism occurs at the level of transcription and involves the cyclic AMP receptor protein. The repressing system is saturated at lower concentrations of cyclic AMP than is the positive control system.
Introduction and expression of a rabbit beta-globin gene in mouse fibroblasts.
Authors: Wold B , Wigler M , Lacy E , Maniatis T , Silverstein S , Axel R
Published: May 2006

The cloned chromosomal rabbit beta-globin gene has been introduced into mouse fibroblasts by DNA-mediated gene transfer (transformation). In this report, we examine the expression of the rabbit gene in six independent transformants that contain from 1 to 20 copies of the cloned globin gene. Rabbit globin transcripts were detected in two of these transformants at steady-state concentrations of 5 and 2 copies per cell. The globin transcripts from one cell line are polyadenylylated and migrate as 9S RNA on methylmercury gels. These transcripts reflect correct processing of the two intervening sequences but lack 48 +/- 5 nucleotides present at the 5' terminus of rabbit erythrocyte globin mRNA.
Structural characterization of the murine fourth component of complement and sex-limited protein and their precursors: Evidence for two loci in the
S
region of the
H-2
complex
Authors: Parker Keith L. , Roos Marleen H. , Shreffler Donald C.
Published: May 2006

The
S
region of the murine major histocompatibility complex controls the expression of two related, serum substance-positive proteins; one (C4) has functional complement activity, whereas the other, the sex-limited protein (Slp), is hemolytically nonfunctional. The structural relationships of these molecules to each other and to their putative intracellular precursors have been examined. Radiolabeled intracellular C4 and Slp precursors were isolated from lysates of cultured peritoneal cells. The C4 and Slp precursors and their processed subunits were purified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Antigenically distinct precursors for C4 and Slp were demonstrated by sequential immunoprecipitation experiments in which anti-Slp-reactive molecules were precleared by exhaustive immunoprecipitation and residual C4 molecules were precipitated by antibody to serum substance. Both molecules had apparent molecular weights of 185,000. Their molecular identities as precursors of the mature C4 and Slp proteins were established in pulse-chase studies and by comparisons of their tryptic peptide profiles with those of isolated subunits from the processed proteins. When isolated α- or β-subunits from C4 and Slp proteins were compared by peptide mapping, it was possible to detect multiple distinct and multiple shared peptides. This evidence indicates that the C4 and Slp proteins derive from distinct precursor polypeptides and suggests that the primary structures of the C4 and Slp α- and β-subunits are different. These results support the postulate that the
S
region contains two discrete structural loci that specify discrete C4 and Slp proteins.
Reactivation of herpes simplex virus type 2 from a quiescent state by human cytomegalovirus.
Authors: Colberg-Poley A M , Isom H C , Rapp F
Published: May 2006

The ability of human cytomegalovirus to stimulate replication of herpes simplex virus type 2 (HSV-2) was examined. The system used involved HSV-2-infected human embryonic lung cells under conditions (39.5-40 degrees C) in which HSV-2 remains undetectable. Reactivation of HSV-2 was maximal and persisted for the longest duration when cultures were superinfected with 0.02 plaque-forming unit of human cytomegalovirus per cell. Infectious HSV-2 appeared 2 days after superinfection with human cytomegalovirus and ranged from 10(2) to 10(6) plaque-forming units per culture. Virus reactivated from these cultures was neutralized by rabbit immune serum produced against HSV-2. The specificity of this interaction was demonstrated by various criteria: production of HSV-2 was not observed in cultures treated with mock infecting fluid, and inactivation of human cytomegalovirus by heat, ultraviolet irradiation, or immune serum prior to superinfection eliminated its ability to induce HSV-2 replication. These results sugges that interaction between these two human herpesviruses may be of importance in herpesvirus latency in vivo.
Specific association of neurotransmitter with somatic lysosomes in an identified serotonergic neuron of Aplysia californica.
Authors: Schwartz J H , Shkolnik L J , Goldberg D J
Published: May 2006

Injection of [3H]serotonin directly into the cell body of the giant metacerebral neuron (GCN), an identified serotonergic cell in the Aplysia cerebral ganglion, revealed a striking association of the labeled transmitter with large lysosomes. Most of the [3H]serotonin in the neuron is sequestered by lysosomes, which may function as storage depots for the transmitter. This localization is specific: [3H]serotonin was not found in similar lysosomes in the perikaryon of R2, an identified Aplysia cholinergic neuron. [3H]Dopamine, [3H]histamine, and [3H]-N-acetylgalactosamine injected into the serotonergic neuron were not localized in lysosomes. Because of this specificity, it may be possible to use lysosomal binding of labeled transmitter as a radioautographic method for identifying the transmitter type of a nerve cell body. These organelles are lysosomal in origin because they contain acid phosphatase activity. Moreover, examination of isolated GCN cell bodies by fluorescence microscopy revealed the presence of abundant autofluorescent yellow pigment characteristic of lipofuscin. We suggest that [3H]serotonin labels organelles containing membranes that once were integral components of the serotonergic vesicle. Lysosomes might accumulate the transmitter with a high degree of specificity because they contain membranes, perhaps recycled from the neuron's synaptic terminals, that retain their ability to concentrate and bind serotonin.
Protein and phospholipid methylation during chemotaxis in Dictyostelium discoideum and its relationship to calcium movements.
Authors: Mato J M , Marín-Cao D
Published: May 2006

Suspensions of cyclic AMP sensitive cells of Dictyostelium discoideum responded to a cyclic AMP pulse with increased methylation of a protein of molecular weight about 120,000 and increased phospholipid demethylation. Protein methylation reached its peak 15-30 sec after cyclic AMP addition. Phospholipid demethylation reached its maximum within 2 min and basal levels were recovered in 3 min. S-Adenosyl-L-methionine is probably the methyl donor. In vitro addition of 0.25 mM and 25 microM S-adenosyl-L-methionine to sonicated D. discoideum cells inhibited ATP-dependent 45Ca2+ uptake by 70% and 25%, respectively. Based on these lines of evidence we propose that protein and phospholipid methylation are involved in D. discoideum chemotaxis probably by regulation of intracellular Ca2+ movements.
Semisynthesis of human somatotropin analogs.
Authors: Li C H , Blake J
Published: May 2006

Complementation of the natural NH2-terminal 134-amino acid fragment of the reduced, carbamoylmethylated human somatotropin with synthetic analogs of COOH-terminal fragments of 57, 42, or 38 amino acids of reduced, carbamoyl-methylated human somatotropin has been investigated. It was found that synthetic fragments of 57 and 42 amino acids gave recombinants with full growth-promoting activity, whereas attempts to obtain a recombinant with the synthetic 38-amino acid fragment were unsuccessful. The synthesis of two analogs of the COOH-terminal fragment of human somatotropin, human [N]e170, Ala165,182,189] somatotropin-(150-191) and human [Nle170, Ala165,182,189] somatotropin-(154-191), is herein described.
Identification of the lexA gene product of Escherichia coli K-12.
Authors: Little J W , Harper J E
Published: May 2006

The Escherichia coli lexA gene encodes a product important in induction of the recA gene and the expression of various cellular functions, including mutagenesis and prophage induction. As a start in a biochemical analysis of the lexA function, a family of lambda transducing phages carrying lexA+, lexA3, lexA3 spr-54, and lexA3 spr-55 alleles of the lexA gene was isolated and characterized. Polypeptides synthesized by these phages were examined. lambdalexA+ made a distinctive protein 24 kilodaltons (kd) in size. Lambda lexA3, which encodes an active mutant form of the protein dominant to wild-type function, made a slightly larger protein 25 kd in size. The latter protein was shown to be the mutant lexA3 gene product by the fact that lambda lexA3 spr-55, which carries an amber mutation in lexA3, made the 25-kd protein in hosts with an amber suppressor but not in a suppressor-free host. In hosts carrying a multicopy lexA3 plasmid, neither the 25-kd nor the 24-kd protein was made. This result suggests that lexA is autoregulated and that expression of the 24-kd protein made by lambda lexA+ is subject to the same controls. This and other evidence argues that the 24-kd protein is the product of the wild-type lexA+ gene.
Processing in vitro of placental peptide hormones by smooth microsomes.
Authors: Bielinska M , Rogers G , Rucinsky T , Boime I
Published: May 2006

Rough and smooth microsomes were prepared from ascites tumor cells, rat liver, and bovine adrenal cortex. Proteolytic removal of the signal peptide in pre-placental lactogen and asparagine-linked glycosylation of the alpha subunit of chorionic gonadotropin by these fractions were examined in mRNA-dependent lysates from ascites cells. Both processing steps were performed by smooth microsomes, which was unexpected because it has been presumed that only rough microsomes contain components for ribosomal binding. Thus smooth microsomes are apparently capable of interacting with polysomes bearing secretory nascent chains, and cleavage and asparagine-linked glycosylation activities are present in both rough and smooth endoplasmic reticulum.
Prostaglandin D2, a neuromodulator.
Authors: Shimizu T , Mizuno N , Amano T , Hayaishi O
Published: May 2006

The distribution of prostaglandin D synthetase activity was determined in various tissues of rat by using the supernatant fraction (10,000 x g, 20 min) of the homogenates. The highest activity was found in brain, spinal cord, and alimentary tract. The activity was uniquitously distributed in all parts of brain, and the highest specific activity was found in hypothalamus and thalamus. Homogenates of two neuroblastoma cell lines were found to produce prostaglandin D2, whereas a glioma cell line was almost inactive. Prostaglandin D2 is a potent and specific activator of the adenylate cyclase system of cultured neuroblastoma cells, suggesting the possibility that it may act as a neuromodulator in the central nervous system.
Proteases in cellular slime mold development: evidence for their involvement.
Authors: Fong D , Bonner J T
Published: May 2006

Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation.
Human heterozygosity: a new estimate.
Authors: McConkey E H , Taylor B J , Phan D
Published: May 2006

Several hundred polypeptides from four human diploid fibroblast cell lines were compared by high-resolution two-dimensional polyacrylamide gel electrophoresis and double-label autoradiography under conditions where allelic products that differ by a single charged amino acid would be distinguished. The average heterozygosity represented by this set of gene products appears to be less than 1% for changes involving charged amino acids.
Synthesis and assembly of membrane glycoproteins: presence of leader peptide in nonglycosylated precursor of membrane glycoprotein of vesicular stomatitis virus.
Authors: Irving R A , Toneguzzo F , Rhee S H , Hofmann T , Ghosh H P
Published: May 2006

Translation of mRNA encoding vesicular stomatitis virus envelope glycoprotein G by as membrane-free ribosomal extract obtained from HeLa cells yielded a nonglycosylated protein (G1 (Mr 63,000). In the presence of added microsomal membranes, G1 was converted to the glycosylated protein (G2 (Mr 67,000) which is inserted in the membrane vesicles as a transmembrane protein. Labeling with methionine donated by wheat germ initiator tRNA1Met showed that G1 but not G2 contains methionine in the NH2-terminal position. Determination of the NH2-terminal sequence of G1, G2, and G showed that a leader peptide of 16 amino acids is present in G1 but absent from the glycosylated proteins G2 and G. This leader peptide contains at least 62% hydrophobic amino acids and is removed presumably during insertion of G1 into the membrane.
Nucleotide sequence of Escherichia coli K-12 replication origin.
Authors: Sugimoto K , Oka A , Sugisaki H , Takanami M , Nishimura A , Yasuda Y , Hirota Y
Published: May 2006

From subfragments of an EcoRI fragment (9 kilobase pairs) that contained the replication origin of the Escherichia coli chromosome and had been cloned as a recombinant with a nonreplicating DNA fragment coding for ampicillin resistance, small derivative plasmids were constructed. The smallest of these, pTSO151, contained a segment of 463 base pairs as the chromosomal component. Another plasmid, pSY134, constructed from BamHI digests of the EcoRI fragment and mini-F(pMF21), contained a region of 422 base pairs identical with a corresponding region in pTSO151. We conclude that the replication origin of E. coli chromosome is located within this 422-base-pair segment. The nucleotide sequence of this segment is presented.
Nucleotide sequence of the origin of replication of the Escherichia coli K-12 chromosome.
Authors: Meijer M , Beck E , Hansen F G , Bergmans H E , Messer W , von Meyenburg K , Schaller H
Published: May 2006

The origin of replication, oriC, of the Escherichia coli chromosome was mapped within a DNA segment of 422 base pairs. The nucleotide sequence of this segment was determined. The source of DNA for the sequence analysis was a minichromosome constructed in vivo, consisting exclusively of chromosomal DNA and a minichromosome constructed by cloning in vitro. The nucleotide sequence of the replication origin is characterized by a high degree of repetitiveness due to both inverted and direct repeats. Sequence homologies were found between portions of the replication origins of E. coli and phages lambda and G4. This suggests similarities in some steps in the initiation of replication of the different replicons.
Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin.
Authors: Weisbrod S , Weintraub H
Published: May 2006

The globin gene is preferentially sensitive to digestion by DNase I in erythrocyte chromatin but not in brain, fibroblast, or oviduct chromatin. Elution of the erythrocyte chromatin with 0.35 M NaCl leads to no detectable change in the gross structure of individual nucleosomes; however, in this depleted chromatin the globin gene is no longer preferentially sensitive to DNase I. Reconstitution of the depleted chromatin with either the entire 0.35 M NaCl fraction or a subclass from this fraction greatly enriched in two high mobility group proteins (nos. 14 and 17) results in the successful reconstitution of DNase I sensitivity of the globin gene. For all of these preparations, the inactive ovalbumin gene exhibited no preferential sensitivity to DNase I. Reconstitution of the erythrocyte 0.35 M NaCl fraction with depleted brain chromatin resulted in no preferential sensitivity of the globin gene in brain chromatin; however, reconstitution of the brain 0.35 M NaCl fraction with depleted erythrocyte chromatin led to successful reconstitution of DNase I sensitivity of the globin gene. Thus, the eluted proteins responsible for conferring DNase I sensitivity are probably not tissue-specific and probably do not recognize specific DNA sequences.
Vasoactive intestinal polypeptide: specific binding to rat brain membranes.
Authors: Taylor D P , Pert C B
Published: May 2006

The binding of radiolabeled vasoactive intestinal polypeptide (VIP) to rat brain membranes was investigated. Specific binding of 125I-labeled VIP was reversible and saturable (Bmax = 2.2 pmol/g of wet tissue). Brain membranes exhibited a high affinity for 125I-labeled VIP (KD = 1 nM) at a single class of noninteracting sites. Binding of 125I-labeled VIP paralleled its immunohistochemical localization, being enriched in cerebral cortex, hippocampus, striatum, and thalamus, with the notable exception of the hypothalamus, which had low levels of binding. The density of sites was greater in synaptosomal fractions relative to mitochondrial or nuclear fractions. Secretin and partial sequences of it and VIP inhibited binding to brain membranes with an order of potency similar to that found in other systems. The findings suggest the existence of a unique new class of brain receptors.
Cyclic AMP-dependent ATPase activity of bovine heart protein kinase.
Authors: Armstrong R N , Kondo H , Kaiser E T
Published: May 2006

The adenosine 3",5"-monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase and protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgADP) values with thermodynamic dissociation constants determined by equilibrium dialysis. The hydrolysis of ATP is stimulated 10- to 20-fold by cAMP in the holoenzyme. The molar specific activity of the catalytic subunit ATPase is approximately 0.7 min-1 with Km(MgATP) = 5 muM. MgADP is a competitive inhibitor of the reaction with a Ki value of approximately muM. The order of the relative effectiveness of metal ions for both ATPase and peptide kinase activities is Mg2+ greater than Mn2+ greater than Ca2+. A possible interpretation of these observations is that the role that the metal ion plays is more directly manifested in bond-breaking than in bond-forming.
Rapid sequence determination of late simian virus 40 16S mRNA leader by using inhibitors of reverse transcriptase.
Authors: Bina-Stein M , Thoren M , Salzman N , Thomspon J A
Published: May 2006

A method for the determination of the primary structure of spliced mRNA junction and leader sequences is described. By analogy to the DNA sequencing procedure of Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci USA 74, 5463--5467], we use 2",3'-dideoxynucleoside triphosphates as chain-terminating inhibitors of the reverse transcriptase (RNA-dependent DNA polymerase) reaction. By using specific DNA restriction fragments as primers in combination with this technique, we have determined the sequence of the spliced junction between the body and the leader sequence of the 16S late mRNA of simian virus 40. The method described should be of general utility in mapping spliced mRNA regions for which the corresponding protein sequence (if any) is unknown.
Dependence of thermal responses of seeds on membrane transitions
Authors: Hendricks Sterling B. , Taylorson Ray B.
Published: May 2006

Transitions at 28-32°C in membranes of many kinds of seeds influence their germination and subsequent growth. Changes at 28-32°C in the rates of loss of endogenous amino acids from imbibed
Barbarea verna
and
Lactuca sativa
seeds indicate permeation of the plasma membrane. A transition in the same temperature range is shown by the change in fluorescence with temperature of substituted naphthalenesulfonate probe molecules associated with membrane fragments isolated from the seeds. Some other plant responses that appear to be causally connected with the membrane(s) transition(s) in the 28-32°C region are growth of maize seedlings and tomato roots, flowering of some bulbs, and control of seed and fern spore germinations by light. Generality of the control is indicated.
Isolation and analysis of chemosensory behavior mutants in Drosophila melanogaster.
Authors: Tompkins L , Cardosa M J , White F V , Sanders T G
Published: May 2006

A behavioral countercurrent paradigm has been developed for assaying the chemotactic responses of wild-type and mutant Drosophila melanogaster adults. Oregon R males avoid both quinine sulfate and NaCl, whereas Oregon R females reject the quinine salt but are attracted to NaCl when tested in this paradigm. Wild-type behavior is sufficiently reproducible to allow identification of mutants affecting chemotaxis, and 12 such mutants, in six complementation groups, have now been isolated. Three of the mutants respond abnormally to NaCl, two in one complementation group with atactic behavior (no chemotaxis) and the other, in a separate group, with a mistactic response (attraction to the stimulus). Four mutants in another group respond mistactically to quinine sulfate. Of the remaining mutants, two in one group behave atactically and three, in two groups, respond mistactically to either chemical stimulus. Several of the mutants also show abnormal behavior in a proboscis extension assay when tested individually with sucrose solutions.
Transformation of DBA/2 mouse fetal liver cells infected in vitro by the anemic strain of Friend leukemia virus.
Authors: Golde D W , Bersch N , Friend C , Tsuei D , Marovitz W
Published: May 2006

Fetal liver cells of DBA/2 mice were infected with the anemic strain of Friend leukemia virus (FLV-A), which has no spleen focus-forming virus (SFFV) activity. The infected cells were grown in medium with or without erythropoietin. Transformed lines were isolated only from the infected cultures that had been treated with erythropoietin at the time of their initiation. The properties of three permanent cell lines in serial passage for over 2 years are described. Each has an aneuploid karyotype. Only the immature hematopoietic cells of the first line have metacentric chromosomes. They grow in suspension, as do the erythroleukemic lines derived from leukemic spleens of FLV-infected mice, and clone on agar. They produce tumors resembling reticulum cell sarcomas upon subcutaneous inoculation into syngeneic hosts. Stimulation of differentiation induced after treatment with dimethyl sulfoxide identifies the cells of the first line as being erythroid in origin. The two other lines are adherent and epithelioid in appearance. These lines may have originated from the nonhematopoietic cells present in fetal liver. No tumors were produced after the subcutaneous inoculation of 10(6) cells. All three lines synthesize virus. The virus is attenuated for leukemogenicity and has no SFFV activity. The transforming event appears to be specific, because fetal liver cells from C57BL/6 mice, which are resistant to the induction of leukemia by FLV, were not affected by the virus. Malignant transformation of erythroid cells by FLV-A in vitro confirms the in vivo findings that SFFV may not be a necessary prerequisite for the induction of erythroleukemia in susceptible hosts.
Interferon induction: a conformational hypothesis.
Authors: Miles D L , Miles D W , Eyring H
Published: May 2006

The ability of polynucleotides or polynucleotide duplexes such as poly(I).poly(C) to induce interferon production is proposed to depend on the existence of certain stable glycosidic orientations. It appears that a slight increase in instability of 1--3 kcal/mole (1 cal = 4.184 J) in the conformational regions near 20 degrees, 80 degrees, and 160 degrees leads to a loss of potency with respect to interferon induction. Thus, it is proposed that, for a polynucleotide to exist in the overall conformation necessary for interferon induction, stability of glycosidic orientations near 20 degrees, 80 degrees, and 160 degrees may be necessary to confer flexibility and activity on polynucleotide structures. This proposed conformational triad of stable conformational regions essential to interferon induction is based on the results of conformational energy calculations of the glycoside rotational profiles of adenosine, 7-deazaadenosine, inosine, and 7-deazainosine, as well as the conformational properties of other purine nucleoside analogs, and on inferences derived from calculations about the conformational effect in polynucleotides of removing the 2'-OH group.