bluuebunny/crossref_metadata_2025 huggingface dataset:
Restoration of normal appearance, growth behavior, and calcium content to transformed 3T3 cells by magnesium deprivation.
Authors: Rubin H , Vidair C , Sanui H
Published: May 2006

A spontaneously transformed clone was isolated from repeatedly passaged BALB/c 3T3 cells. The transformed cells were rounded or slender and elongated, were randomly arranged in an overlapping pattern, grew to high cell density, and had a low requirement for serum. The rates of multiplication and DNA synthesis of the nontransformed and the transformed lines were reduced for several days by drastic reduction in the Mg2+ concentration of the medium, but the rate of DNA synthesis in the Mg2+-deprived cultured increased after 6--8 days, suggesting an adaptation of the cells or a change in local environment. When maintained in very low Mg2+ concentrations the transformed cells assumed the appearance and arrangement of nontransformed cells within 1 day. The rate of DNA synthesis in the transformed cultures in 1.0 mM Mg2+ was independent of serum concentration. After 3 days of Mg2+ deprivation, however, the rate of DNA synthesis became highly dependent on both serum concentration and population density, thus resembling the growth behavior of nontransformed cells. Neither deprivation of K+ or Ca2+ nor addition of dibutyryl cyclic AMP produced these effects. The Mg2+ contents of nontransformed and transformed cells in physiological concentrations of Mg2+ were similar and only slightly reduced by incubation for 4 days in Mg2+-deficient medium. In 1.0 mM Mg2+, the Ca2+ content of the nontransformed cells was approximately 3 times higher than that of the transformed cells. After incubation in Mg2+-deficient medium, the Ca2+ contents of both cells types increased; that of the transformed cells slightly exceeded that of the nontransformed cells in Mg2+-sufficient medium. The results show that Mg2+-deprived transformed cells closely resemble nontransformed cells in appearance, requirement for serum, response to cell population density, and Ca2+ content. They also show that these parameters can be regulated coordinately by Mg2+ and support the suggestion that a defect in regulation by Mg2+ is a basic feature of the malignant transformation.
Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast.
Authors: Rose M , Casadaban M J , Botstein D
Published: May 2006

A plasmid was constructed that allows the selection in vivo of gene fusions between the Escherichia coli beta-galactosidase gene and the yeast (Saccharomyces cerevisiae) URA3 gene. A large yeast DNA fragment containing the URA3 gene was placed upstream of an amino-terminally deleted version of the lacZ gene. The plasmid vehicle contains sequences that allow selection and maintenance of the plasmid in both yeast and E. coli. Selection for Lac+ in E. coli yielded numerous deletions that fused the lacZ gene to the URA3 gene and flanking yeast sequences, to the bacterial tetracycline-resistance gene from the parent plasmid pBR322, and to the yeast 2-micrometer plasmid DNA. Some of these fusion plasmids produced beta-galactosidase activity when introduced into yeast. One of the fusions to the URA3 gene itself has been shown to place the expression of beta-galactosidase activity under uracil regulation in yeasts.
Abnormal regulation of monocyte insulin-binding affinity after glucose ingestion in patients with myotonic dystrophy.
Authors: Moxley R T , Livingston J N , Lockwood D H , Griggs R C , Hill R L
Published: May 2006

Insulin insensitivity of uncertain etiology often exists in myotonic muscular dystrophy, a multitissue, autosomal dominant disorder hypothesized to be a hereditary membrane disease. The present studies show that monocytes from patients with myotonic dystrophy fail to demonstrate the normally observed qualitative increase in insulin-binding affinity after oral glucose loading. Monocytes from 10 normal volunteers developed a significantly increased insulin-binding affinity by 2 hr after glucose ingestion (mean +/- SEM, 11.7 +/- 2.7 ng/ml compared to basal 50% insulin displacement value of 23.3 +/- 2.2 ng/ml, P less than 0.005). This increase was maintained at 5 hr (13.5 +/- 2.7 ng/ml, P less than 0.05). In contrast, no significant increase in monocyte insulin-binding affinity occurred in cells from nine myotonic dystrophy patients at 2 and 5 hr after glucose loading (50% insulin displacement values: basal, 14.2 +/- 2.8 ng/ml; 2 hr, 16.7 +/- 1.7 ng/ml; 5 hr, 10.8 +/- 2.1 ng/ml). These alterations document the presence of abnormalities in the insulin receptor or receptor-associated processes that modulate insulin binding. A hereditary plasma membrane defect may underlie these findings. This abnormality may have an etiologic role in the decreased insulin sensitivity that frequently afflicts patients with myotonic dystrophy.
A third class of avian sarcoma viruses, defined by related transformation-specific proteins of Yamaguchi 73 and Esh sarcoma viruses.
Authors: Ghysdael J , Neil J C , Vogt P K
Published: May 2006

The gag-linked transformation-specific protein (polyprotein) p80 of Esh avian sarcoma virus (ESV) has been compared by tryptic peptide mapping with the homologous protein p90 of Yamaguchi 73 avian sarcoma virus (Y73). p80 of ESV and p90 of Y73 were found to share all four of their major nonstructural, transformation-specific, methionine-containing peptides and to have at least seven cysteine-containing transformation-specific peptides in common. Two nonstructural cysteine-containing peptides unique for ESV p80 and three specific for Y73 p90 were also identified. None of these peptides were found in the transforming gene product pp60src of Rous sarcoma virus (RSV) or in the transformation-specific polyproteins p105 of avian sarcoma virus PRCII (PRCII) or p140 of Fujinami sarcoma virus (FSV). ESV p80 and Y73 p90 are phosphorylated, and their tryptic phosphopeptides appear to be identical. In each polyprotein two major phosphopeptides were demonstrated, one containing phosphoserine, the other phosphotyrosine. The latter serves as phosphoacceptor for the protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) associated with p80 and p90. These protein kinase activities were found to be functionally indistinguishable but could be easily distinguished from the activities associated with PRCII p105 and FSV p140 on the basis of their cation requirement and target site specificity. On that basis also, p80/p90-associated protein kinases were found to be more similar to the enzymatic activity of pp60src than to those associated with the PRCII and FSV transformation-specific polyproteins. These results document a close genetic relationship between the two independently isolated sarcoma viruses Y73 and ESV. On the basis of the relatedness of transformation-specific proteins, ESV and Y73 constitute class III of avian sarcoma viruses, with class I containing the various strains of RSV and class II encompassing FSV and PRCII.
Resolution of dopamine and serotonin receptor components of [3H]spiperone binding to rat brain regions.
Authors: List S J , Seeman P
Published: May 2006

A procedure was developed to identify receptors for dopamine and serotonin separately and selectively by means of [3H]spiperone and to measure the density of each receptor in different regions of the rat brain. In the striatum, the binding of [3H]spiperone to dopamine receptors was inhibited by sulpiride but not by quinazolinedone R43448 (R43448); in the frontal cortex, however, the binding of [3H]spiperone to serotonin receptors was inhibited by R43448 but not by sulpiride. Thus, the density of dopamine receptors (D2 sites) was measured by [3H]spiperone binding in the presence of 0.1 microM R43448 (to preclude the attachment of the 3H-labeled ligand to serotonin sites), while the density of serotonin receptors (S2 sites) was measured by [3H]spiperone binding in the presence of 10 microM sulpiride (to preclude the attachment of the 3H-labeled ligand to dopamine sites). The density of D2 sites was highest in the striatum, followed by the olfactory tubercle, hypothalamus, substantia nigra, and the lower pons--medulla region. All five regions had similar dissociation constants (Kd values) of 0.05--0.15 nM. The density of S2 sites was highest in the frontal cortex, followed by the posterior cortex, olfactory tubercle, striatum, hypothalamus, and thalamus, and all regions had Kd values in the range 0.6--2.3 nM. Thus, because the Kd values were similar for all regions, and because Scatchard analyses revealed a single set of sites for either D2 or S2 (where detected), the main criteria for resolving the dopamine and serotonin components of [3H]spiperone binding were considered fulfilled.
Distribution of proviral sequences in chromatin of embryonic fibroblasts infected by Rous sarcoma virus.
Authors: Pantazis P , Schulz R A , Chirikjian J G , Papas T S
Published: May 2006

Sheared chromatin prepared from chicken embryo fibroblasts and fibroblasts transformed by exogenous Rous sarcoma virus (Schmidt--Ruppin strain D) was separated by rate sedimentation on glycerol gradients into two components: fast-migrating (heavy chromatin fraction) and slow-migrating (light chromatin fraction). DNAs extracted from these fractions were assayed for proviral sequences by molecular hybridization using DNA complementary to the viral sequences. In uninfected cells, the endogenous complementary sequences were found to be equally distributed between heavy and light fractions. However, the newly integrated exogenous proviral sequences were found mostly in the light chromatin fraction in the transformed cells. Additionally, the light fraction was more sensitive to DNase I digestion and contains more material melting at low temperatures when compared with the heavy fraction. The results show that (i) distribution of endogenous proviral sequences is independent of chromatin conformation, and (ii) most of the newly acquired exogenous sequences are integrated within the host's chromatin fraction that exhibits properties of euchromatin. Because chromatin fragmentation and fractionation is accomplished without digestion with degrading enzymes, the chromatin fractions enriched in exogenous sequences remain intact and thus are suitable for further studies.
Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle.
Authors: Umeda P K , Sinha A M , Jakovcic S , Merten S , Hsu H J , Subramanian K N , Zak R , Rabinowitz M
Published: May 2006

Recombinant DNA clones containing sequences for two different types of myosin heavy chain (HC) genes from chicken embryonic skeletal muscle were constructed and analyzed. Specificity of the clones for myosin HC was demonstrated by hybrid-arrested translation, by hybridization to a 7.0-kb mRNA, and by comparison of DNA sequences with known amino acid sequences of rabbit skeletal muscle myosin HC. Restriction enzyme and electron-microscopic heteroduplex analysis showed the presence of two distinct but homologous cDNA sequences. Hybrid melting curves indicated that both types of sequences represent fast myosin HC sequences.
Histone genes are clustered but not tandemly repeated in the chicken genome.
Authors: Engel J D , Dodgson J B
Published: May 2006

The recombinant chicken DNA library was screened for histone genes by using pSp17, a recombinant sea urchin DNA probe containing the H2a and H3 genes of Strongylocentrotus purpuratus. Three of the isolated lambda recombinants have been analyzed by restriction enzyme mapping and Southern blotting; one histone H3 gene-encoding recombinant was further analyzed by DNA sequence determination. These studies reveal that the chicken histone genes are not tandemly reiterated, but that, at the least, several histone genes are physically closely linked in a nonrepetitive arrangement within the chicken genome. The evolutionary implications of this arrangement versus that seen in Drosophila and sea urchins is discussed.
Anchorage-independent growth of normal human fibroblasts.
Authors: Peehl D M , Stanbridge E J
Published: May 2006

Normal human fibroblasts, considered to be entirely anchorage dependent for proliferation, have been grown in methylcellulose medium. The most important factor required for growth in suspension appears to be the use of high levels of serum and hydrocortisone. Newborn foreskin or fetal lung fibroblasts form colonies as large as 0.5 mm in diameter after 3 wk, with a colony-forming efficiency as high as 70%. Mouse 3T3 cells that do not form colonies in standard assays for anchorage-independent growth also grow under these conditions. Colony formation results after inoculation of as few as 100 cells per 60-mm dish, and metaphase cells have been visualized with a fluorescent DNA stain, showing that colony formation is due to division rather than aggregation. Fibroblasts recovered from suspension and grown as monolayers retain a diploid karyotype and normal shape, do not form tumors upon injection into nude mice, and become senescent. Thus, the trait of anchorage-independent growth in vitro is clearly possessed by normal human fibroblasts and can be expressed under the proper conditions.
Leukocyte and fibroblast interferon genes are located on human chromosome 9.
Authors: Owerbach D , Rutter W J , Shows T B , Gray P , Goeddel D V , Lawn R M
Published: May 2006

At least eight leukocyte interferon genes (IFL) and the single fibroblast interferon gene (IFF) have been located on chromosome 9 in humans. In somatic cell hybrids of human and mouse cells containing a normal complement of mouse parental cell chromosomes but reduced numbers of human chromosomes, the human leukocyte and fibroblast interferon DNA sequences were present only when human chromosome 9 was also present.
Contractile activities of structural analogs of leukotrienes C and D: necessity of a hydrophobic region.
Authors: Drazen J M , Lewis R A , Austen K F , Toda M , Brion F , Marfat A , Corey E J
Published: May 2006

Sixteen structural analogs of leukotrienes C and D were tested for their contractile activities on guinea pig pulmonary parenchymal strip and ileum. The analogs differed from the native structures in the position of either the thioether-linked peptide side chain or the hydroxyl group (or both) or in the number and positions of ethylenic bonds. Analogs in which the thioether-linked peptide chain was attached other than at the C-6 position had substantial reductions in activity on both smooth muscle preparations, whereas analogs in which the various ethylenic bonds were saturated retained substantial contractile activity in both assays. These observations demonstrate that, although a hydrophobic region of the eicosinoid is necessary for contractile activity, the length of this segment is more critical than its precise stereochemistry. Analogs of leukotrienes based on the possibility of parallel biosynthetic routes deriving from 8-, 11-, and 15-hydroperoxyeicosatetraenoic acid as precursors were found to effect a comparatively weak contractile response so that their role as biological agents in this respect seems unlikely.
Nitration of naphthalene and remarks on the mechanism of electrophilic aromatic nitration
Authors: Olah George A. , Narang Subhash C. , Olah Judith A.
Published: May 2006

Naphthalene was nitrated with a variety of nitrating agents. Comparison of data with Perrin's electrochemical nitration [Perrin, C. L. (1977)
J. Am. Chem. Soc.
99, 5516-5518] shows that nitration of naphthalene gives an α-nitronaphthalene to β-nitronaphthalene ratio that varies between 9 and 29 and is thus not constant. Perrin's data, therefore, are considered to be inconclusive evidence for the proposed one-electron transfer mechanism for the nitration of naphthalene and other reactive aromatics. Moodie and Schoefield [Hoggett, J. G., Moodie, R. B., Penton, J. R. & Schoefield, K. (1971)
Nitration and Aromatic Reactivity
(Cambridge Univ. Press, London)], as well as Perrin, independently concluded that, in the general scheme of nitration of reactive aromatics, there is the necessity to introduce into the classical Ingold mechanism an additional step involving a distinct intermediate preceding the formation of the Wheland intermediate (σ complexes). This view coincides with our two-step mechanistic picture [Kuhn, S. J. & Olah, G. A. (1961)
J. Am. Chem. Soc.
83, 4564-4571] of the nitronium salt nitration of aromatic hydrocarbons (including benzene and toluene), in which low substrate selectivity but high positional selectivity was found, indicating the independence of substrate from positional selectivity.
Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus.
Authors: DeFeo D , Gonda M A , Young H A , Chang E H , Lowy D R , Scolnick E M , Ellis R W
Published: May 2006

Harvey murine sarcoma virus (Ha-MuSV) is a mouse-rat recombinant retrovirus that encodes a protein designated p21, required for virally induced transformation. Using a radiolabeled DNA fragment from the p21 coding region, we have detected homologous DNA sequences in the normal DNA of rats and of several other vertebrate species. Moreover, many tested cells from these species contain low levels of a p21 protein that is highly related to viral 21. Now we report two independent fragments from normal rat DNA containing sequences (sarc) homologous to the Ha-MuSV transforming region that were cloned in the bacteriophage vector Charon 4A. Sarc sequences in the one fragment are completely colinear with the viral sequences and share apparently all restriction endonuclease sites. Sarc sequences in the second fragment have several sets of intervening sequences and lack some restriction endonuclease sites found in the viral transforming region. Despite the presence of these intervening sequences in the second sarc fragment, we have been able to ligate this sarc fragment to the long terminal repeat sequence of HaMuSV and to induce cellular transformation and high levels of p21 expression upon transfection of this DNA to NIH 3T3 mouse cells. These results suggest that elevated levels of p21, normally expressed at low levels in a variety of cells, can induce cellular transformation.
Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells.
Authors: Seamon K B , Padgett W , Daly J W
Published: May 2006

The diterpene, forskolin [half-maximal effective concentration (EC50), 5-10 microM] activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in rat cerebral cortical membranes in a rapid and reversible manner. Activation is not dependent on exogenous guanyl nucleotides and is not inhibited by guanosine 5'-O-(2-thiodiphosphate) when assayed with adenosine 5'-[beta, gamma-imido]triphosphate as substrate. GTP and GDP potentiate responses to forskolin. The activations of adenylate cyclase by forskolin and guanosine 5'-[beta, gamma-imido]triphosphate p[NH]ppG are not additive, whereas activations by forskolin and fluoride are additive or partially additive. The responses of adenylate cyclase to forskolin or fluoride are not inhibited by manganese ions, whereas the response to p[NH]ppG is completely blocked. Activation of adenylate cyclase by forskolin is considerably greater than the activation by fluoride in membranes from rat cerebellum, striatum, heart, and liver, while being about equal or less than the activation by fluoride in other tissues. Forskolin (EC50, 25 microM) causes a rapid and readily reversible 35-fold elevation of cyclic AMP in rat cerebral cortical slices that is not blocked by a variety of neurotransmitter antagonists. Low concentrations of forskolin (1 microM) augment the response of cyclic AMP-generating systems in brain slices to norepinephrine, isoproterenol, histamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin would appear to activate adenylate cyclase through a unique mechanism involving both direct activation of the enzyme and facilitation or potentiation of the modulation of enzyme activity by receptors or the guanyl nucleotide-binding subunit, or both.
Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers.
Authors: Boheim G , Hanke W , Barrantes F J , Eibl H , Sakmann B , Fels G , Maelicke A
Published: May 2006

Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer.
Estimation of genetic variation at the DNA level from restriction endonuclease data.
Authors: Ewens W J , Spielman R S , Harris H
Published: May 2006

We consider the estimation of the genetic variation in a natural population when the data are obtained by the use of restriction endonucleases. Under the restriction endonuclease technique, a particular DNA segment is considered and cut wherever a recognition sequence appropriate to the endonuclease occurs. We consider data generated when a random sample of homologous DNA segments is treated in this way with one or a battery of restriction endonucleases. The numbers and sizes of the fragments that result indicate the locations and the frequencies of the recognition sequence (or, with a battery of restriction endonucleases, of each recognition sequence). These frequencies in the sample form the basis for an estimate of the amount of genetic variation in the population.
Isolation of two clusters of mouse histone genes.
Authors: Sittman D B , Chiu I M , Pan C J , Cohn R H , Kedes L H , Marzluff W F
Published: May 2006

Histone mRNA was partially purified from mouse myeloma cells synchronized in S phase by isoleucine starvation. A cDNA was prepared that contained sequences complementary to all five mouse histone genes. This cDNA was used to screen a library of mouse DNA in lambda phage. The positive clones were screened by hybridization with sea urchin histone gene-specific probes to identify those clones that contained histone genes. Confirmation of this identification was obtained by hybridization with Drosophila histone genes. Two independent clusters of histone genes were isolated. One, MM531, contains regions hybridizing specifically to H3, H4, and H1 and the other, MM221, contains two regions hybridizing specifically to H3 and single regions complementary to H4, H2b, and H2a. They are not part of a simple repeating structure. The nucleotide sequence of the coding region of the H3 gene in MM531 has been determined. This gene could code for a variant H3 protein that has several amino acid substitutions not reported in other H3 proteins.
Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts.
Authors: Decker S
Published: May 2006

Protein phosphorylation was examined in whole cell extracts from normal and avian sarcoma virus-transformed chicken embryo fibroblasts. The addition of serum or epidermal growth factor to serum-starved normal cells resulted in increased 32P labeling of a Mr 30,000 protein. In extracts from cells transformed by a temperature-sensitive mutant of Schmidt-Ruppin virus, subgroup A, and grown at the permissive temperature, the protein was phosphorylated regardless of serum starvation. This Mr 30,000 protein was shown to be ribosomal protein S6, and the effects of avian sarcoma virus transformation on S6 phosphorylation were further investigated. The ability to phosphorylate S6 in the absence of serum was found to be temperature sensitive when S6 preparations from the temperature-sensitive mutant-infected cells incubated at permissive and nonpermissive temperatures were compared. Cells transformed by the parent virus (Schmidt-Ruppin, subgroup A) maintained the ability to phosphorylate S6 in the absence of serum when incubated at either temperature. Phosphoserine was the only phospho-amino acid detected in acid hydrolysates from phosphorylated S6 preparations.
Stabilization and the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition.
Authors: Schliwa M , van Blerkom J , Porter K R
Published: May 2006

Treatment of epithelial BSC-1 cells with low concentrations of the detergent Brij 58 results in partial or complete removal of the plasmalemma and partial extraction of internal membrane-bound organelles without causing massive release of "cytosolic" proteins from the cytoplasmic ground substance. Stereoscopic high-voltage electron microscopy of such extracted and fixed cells demonstrates a system of slender (4-20 nm) strands in a three-dimensional "microtrabecular" arrangement similar to that observed in unextracted whole-mount preparations. Extraction of Brij-extracted cells with Triton X-100 dissolves many of the microtrabecular strands, leaving, as a more stable structure, a characteristic cytoskeletal network composed of various filaments and microtubules. Two-dimensional polyacrylamide gel electrophoresis of 35S-labeled polypeptides performed concurrently with the morphological studies demonstrates that Triton extraction of Brij-extracted cells releases a large number of polypeptides. This release parallels the loss of structural components observed by electron microscopy. Labeling of Brij-extracted cells with heavy meromyosin subfragment 1 decorates actin filaments with characteristic arrowhead complexes which are readily visualized only after subsequent Triton extraction. These observations support the concept that many cytoplasmic proteins are structure-bound and, in addition to the components comprising the cytoskeleton, are structure-forming. We conclude that a metastable association of various proteins of the cytoplasmic ground substance exists whose morphological integrity is maintained, at lest temporarily, after removal of the plasmalemma in solutions containing Brij 58.
Inhibition of specific cell-mediated cytotoxicity by monoclonal antibodies to human T cell antigens
Authors: Platsoucas Chris D. , Good Robert A.
Published: May 2006

Human T lymphocyte subpopulations recently have been defined by monoclonal antibodies that recognize cell-surface antigens selectively expressed on functionally distinct T cell subsets. The majority (approximately 90%) of the peripheral blood sheep erythrocyte-rosette-forming cells carry the OKT3 antigen. Helper cells are OKT4
+
, whereas cytotoxic/suppressor cells are OKT5
+
and OKT8
+
. We investigated the effect of several monoclonal antibodies recognizing T cell antigens on certain proliferative responses of T cells and on the effector phase of the specific T cell-mediated cytotoxicity generated in mixed lymphocyte culture (MLC). In the absence of added complement, (
i
) OKT3 and OKT4 monoclonal antibodies inhibited the proliferative response to phytohemagglutinin (PHA), (
ii
) OKT3 monoclonal antibody inhibited the proliferative response to allogeneic cells in MLC, and (
iii
) OKT3 monoclonal antibody significantly and regularly inhibited the effector phase of the specific T cell-mediated cytotoxicity against allogeneic targets (
P
< 0.001) in a concentration-dependent manner. The OKT5 and OKT8 monoclonal antibodies, again in the absence of complement, inhibited moderately the specific cell-mediated cytotoxicity. This inhibition was observed in some experiments only. Inhibition of the specific cytotoxicity by these antibodies also was observed in secondary responses. In contrast, again in the absence of added complement, none of these antibodies had an effect on the nonspecific cytotoxicity generated in MLC against the K562 targets. The OKT4 antibody in the absence of added complement had no effect on either the specific or nonspecific cytotoxicity. Furthermore, treatment with OKT3 or OKT8 antibody and complement completely abrogated the specific T cell-mediated cytotoxicity but had no effect on the natural killer-like cytotoxicity against the K562 cells. Treatment with the OKT4 antibody and complement had no effect. These results suggest that (
i
) the T5/T8 and T3 antigens, present on cytotoxic T lymphocytes, may be involved directly or indirectly in the antigen recognition step(s) or the lytic mechanism of T cell-mediated lympholysis; and (
ii
) nonspecific cytotoxicity against the K562 targets generated in MLC is mediated by cells phenotypically different than those that mediate specific cytotoxicity.
Taxol, a microtubule stabilizing agent, blocks the replication of Trypanosoma cruzi.
Authors: Baum S G , Wittner M , Nadler J P , Horwitz S B , Dennis J E , Schiff P B , Tanowitz H B
Published: May 2006

Taxol, an experimental antitumor agent and stabilizer of microtubules, inhibits in vitro replication of the human pathogenic hemoflagellate Trypanosoma cruzi. Micromolar concentrations of the drug prevent the completion of cell division in these organisms but allow the multiplication of cell organelles such as the nucleus, kinetoplast, and flagellum. The result is the formation of motile organisms that have extra organelles but cannot fully replicate. Division proceeds to a relatively fixed locus on the long axis of the organism, suggesting the presence of a specific affected structure or function at this site. It is postulated that taxol produces these effects by stabilizing a portion of the microtubular cytoskeleton of T. cruzi.
Evidence against extrapancreatic insulin synthesis.
Authors: Eng J , Yalow R S
Published: May 2006

Labeled and unlabeled insulin in acid/ethanol tissue extracts can be concentrated up to 100-fold by using a hydrophobic adsorption technique. After adsorption to and elution from an octadecylsilyl silica column, insulin is recovered in yields greater than 75%. By using this method of concentration, insulin in brain tissues of three of four fed rats and one rabbit was found to be less than 20% of plasma concentration. The kidney is the only extrapancreatic organ in which insulin is observed to be markedly above plasma levels. Porcine-insulin-like material was not detectable in guinea pig tissues (less than 0.02 ng/g). It is concluded that insulin is not synthesized in brain or other extrapancreatic tissues and that other mammalian insulins are not found in guinea pig tissues.
Characterization and partial purification of solubilized active opiate receptors from toad brain.
Authors: Ruegg U T , Cuenod S , Hiller J M , Gioannini T , Howells R D , Simon E J
Published: May 2006

Opiate receptors have been solubilized from toad brain membranes in active form by using digitonin. Between 40% and 50% of the stereospecific binding activity present in toad brain membranes is recoverable in the ultracentrifugal supernatant of digitonin extracts. Binding of opiates to the solubilized receptor is enhanced 4- to 5-fold by decreasing digitonin concentration to 0.1% or less prior to binding. The solubilized receptor is similar to the membrane-bound receptor in its affinity for various ligands and its sensitivity to heat, trypsin, and N-ethylmaleimide. Moreover, the sodium effect seen in membrane-bound receptor is retained in the solubilized preparation. Both membrane-bound and soluble toad receptors show weak binding of enkephalins, suggesting that they are predominantly of the mu type. The solubilized opiate receptor has an approximate molecular weight of 350,000-400,000. Purification of up to 20-fold has been achieved by gel filtration on Sepharose CL-6B.
Anatoxin-a interactions with cholinergic synaptic molecules.
Authors: Aronstam R S , Witkop B
Published: May 2006

Anatoxin-a, a bicyclic amine isolated from blue-green alga, binds to the nicotinic acetylcholine receptor of Torpedo electric tissue, thereby inducing conformational changes in the postsynaptic receptor--ion channel complex as evidenced by alterations in the binding of radiolabeled ligands to the complex. Anatoxin-a binds to the acetylcholine recognition site (Kd = 0.1--0.2 microM) as indicated by its competitive inhibition of specific [3H]acetylcholine and d-[3H]tubocurarine binding, Anatoxin-a stimulates the binding of three physiologically identified "ion channel blockers," [3H]perhydrohistrionicotoxin, [3H]phencyclidine, and [3H]phencyclidine methiodide. The 50% effective doses for these effects range from 0.14 to 0.28 microM. Incubation of Torpedo membranes with anatoxin-a before addition of a radiolabeled channel probe produces a time- and concentration-dependent attenuation of the binding compared to the situation in which anatoxin-a and the probe are added simultaneously. The time course for the elaboration of this decrease corresponds to electrophysiological measurements of anatoxin-a-induced desensitization of neuromuscular junction responses. In these nicotinic actions, anatoxin-a is about as potent as acetylcholine. Anatoxin-a has relatively low affinity for the muscarinic acetylcholine receptors of rat brain, inhibiting 3-[3H]quinuclidinyl benzilate binding (10(-10) M) by 50% at concentrations between 10 and 20 microM. In contrast to classical muscarinic agonists, anatoxin-a displays little regional selectivity in its binding, and its receptor affinity is unaltered by alkylation of the neural membranes with N-ethylmaleimide.
alpha-Fetoprotein and albumin genes are in tandem in the mouse genome.
Authors: Ingram R S , Scott R W , Tilghman S M
Published: May 2006

The urine alpha-fetoprotein (AFP) and serum albumin genes most probably arose in evolution as the consequence of a duplication of a common ancestral gene. They have both been previously mapped to chromosome 5 in the mouse. We now have evidence that these genes are closely linked. By using a unique copy DNA probe derived from previously cloned AFP 5' flanking DNA, a recombinant DNA phage has been isolated, from a bacteriophage DNA library, that contains sequences flanking the 5' end of the AFP gene and the 3' end of the albumin gene. Restriction endonuclease mapping and DNA sequence determination of the recombinant phage and comparison to total genomic DNA confirmed that the genes are in tandem, 13.5 kilobase pairs apart, with the albumin gene to the 5' side of the AFP gene. Thus, they are transcribed from the same strand of DNA.
Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.
Authors: Corpéchot C , Robel P , Axelson M , Sjövall J , Baulieu E E
Published: May 2006

Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, I) sulfate (Ia) has been characterized in the anterior and the posterior parts of the brain of adult male rats. Its level (1.58 +/- 0.14 and 4.89 +/- 1.06 ng/g, mean +/- SD, in anterior and posterior brain, respectively) largely exceeded that of I in brain (0.42 +/- 0.10 and 0.12 +/- 0.03 ng/g in anterior and posterior brain, respectively) and of Ia in plasma (0.26 +/- 0.13 ng/ml). Brain Ia level did not seem to depend on adrenal secretion; it was unchanged after administration of corticotropin or dexamethasone for 3 days, and no meaningful change occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. In contrast, stress conditions prevailing 2 days after adrenalectomy plus orchiectomy or after the corresponding sham operation resulted in a significantly increased concentration of Ia in the brain. Changes of Ia level in brain occurred irrespective of changes in corresponding plasma samples. It is proposed that Ia formation or accumulation (or both) in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system.
Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.
Authors: de Lanerolle P , Adelstein R S , Feramisco J R , Burridge K
Published: May 2006

Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth muscle myosin light chain kinase and there is an inverse relationship between the kinase activity and the amount of antibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, alpha-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to alpha-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.
Antibodies specific for the polyoma virus middle-size tumor antigen.
Authors: Walter G , Hutchinson M A , Hunter T , Eckhart W
Published: May 2006

We have obtained antibodies specific for the polyoma virus middle-size tumor antigen (middle T antigen) by immunizing rabbits with a synthetic peptide, Lys-Arg-Ser-Arg-His-Phe, corresponding to the six carboxy-terminal amino acids of the middle T antigen predicted from the nucleotide sequence of polyoma DNA. The antipeptide serum precipitates the polyoma middle T antigen but not the small or large tumor antigens, and precipitation is inhibited in the presence of the peptide. Two cellular proteins, 30,000 and 26,000 daltons, are also precipitated specifically by the antipeptide serum and may have amino acid sequences related to the peptide. Two other cellular proteins, 33,000 and 25,000 daltons, are precipitated only in the presence of the peptide and may associate with it in cell extracts. Antisera directed against synthetic peptides are likely to be important in various ways, including the production of antibodies directed against particular determinants and the recognition of unknown proteins whose genes have been analyzed.
ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type.
Authors: Jaurin B , Grundström T
Published: May 2006

A 1536-nucleotide-long sequence that carries the ampC beta-lactamase gene of the Escherichia coli K-12 chromosome has been determined. This gene codes for a protein of 377 amino acids, of which the first 19 amino acids form a signal peptide. The molecular weight of the mature enzyme was determined to be 39,600. The ampC beta-lactamase with a substrate specificity for cephalosporins showed no significant sequence homologies with beta-lactamases of the penicillinase type or with D-alanine carboxypeptidases. However, because the region around serine-80 of the ampC beta-lactamase has extensive homology with an active-site fragment of the Pseudomonas aeruginosa cephalosporinase, we suggest that the ampC cephalosporinase as well as related cephalosporinases form a distinct group of serine beta-lactamases that have an evolutionary origin different from that of the serine penicillinases and thus constitute a new class of beta-lactamases.
Transformation-enhancing activity of gelatin-binding fragments of fibronectin.
Authors: De Petro G , Barlati S , Vartio T , Vaheri A
Published: May 2006

Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.
Activation of related transforming genes in mouse and human mammary carcinomas.
Authors: Lane M A , Sainten A , Cooper G M
Published: May 2006

High molecular weight DNAs of five tumors induced by mouse mammary tumor virus (MMTV), two mouse mammary tumors induced by a chemical carcinogen, and one human mammary tumor cell line (MCF-7) were assayed for the presence of transmissible activated transforming genes by transfection of NIH 3T3 mouse cells. DNAs of all five MMTV-induced tumors, one chemical carcinogen-induced tumor, and the human tumor cell line induced transformation with high efficiencies (approximately 0.2 transformant per micrograms of DNA). NIH cells transformed by DNAs of MMTV-induced tumors did not contain exogenous MMTV DNA sequences, indicating that MMTV-induced mammary carcinomas contained activated cellular transforming genes that were not linked to viral DNA. The transforming activities of DNAs of all five MMTV-induced tumors, the chemical carcinogen-induced mouse tumor, and the human tumor cell line were inactivated by digestion with the restriction endonucleases Pvu II and Sac I, but not by BamHI, EcoRI, HindIII, Kpn I, or Xho I. These results indicate that the same or closely related transforming genes were activated in six different mouse mammary carcinomas, induced by either MMTV or a chemical carcinogen, and in a human mammary carcinoma cell line.
Obligately barophilic bacterium from the Mariana trench.
Authors: Yayanos A A , Dietz A S , Van Boxtel R
Published: May 2006

An amphipod (Hirondellea gigas) was retrieved with decompression in an insulated trap from an ocean depth of 10,476 m. Bacterial isolates were obtained from the dead and cold animal by using silica gel medium incubated at 1000 bars (1 bar = 10(5) Pa) and 2 degrees C. The isolate designated MT41 was found to be obligately barophilic and did not grow at a pressure close to that of 380 bars found at average depths of the sea. The optimal generation time of about 25 hr was at 2 degrees C and 690 bars. The generation time at 2 degrees C and 1,035 bars, a pressure close to that at the depth of origin, was about 33 hr. Among the conclusions are: (i) pressure is an important determinant of zonation along the water column of the sea; (ii) some obligately barophilic bacteria survive decompressions; (iii) the pressure of optimal growth at 2 degrees C appears to be less than the pressure at the depth of origin and may be diagnostic for the depth of origin; (iv) rates of reproduction are slow yet significant and an order of magnitude greater than previously thought; and (v) much of deep-sea microbiology may have been done with spurious deep-sea organisms due to warming of samples.
Impact of upwardly revised Δ
H

f

0
of primary, secondary, and tertiary radicals on mechanistic constructs in thermal reorganizations
Authors: Doering William von Eggers
Published: May 2006

Impetus for upward revision in the heats of formation of the ethyl (primary), isopropyl (secondary), and
t
-butyl (tertiary) free radicals has gained sufficient momentum in the last 6 yr to prompt an illustrative assessment of the far-ranging implications for interpretation of thermal reorganizations. Previous obstacles to consideration of diradicals as transition states in rearrangements of cyclopropanes and cyclobutanes are removed. The energetic relation of cyclohexa-1,4-diyl as an intermediate diradical in the Cope rearrangement to the transition state of a concerted mechanism is clarified.
Model of clustering of earthquakes
Authors: Barenblatt G. I. , Keilis-Borok V. I. , Vishik M. M.
Published: May 2006

A two-dimensional model of the expansion of a crack in an elastic medium is considered in which friction depends on the slip rate and the modulus of cohesion depends on the speed of expansion of the crack. Elastic waves are neglected (quasi-static model). Under some conditions, the expansion of the crack is realized by the alternation of slow and fast episodes (“shocks”) of slip. This offers a possible qualitative explanation of several forms of earthquake clustering, including clustering that is premonitory to strong earthquakes.
18S defective interfering RNA of Semliki Forest virus contains a triplicated linear repeat.
Authors: Lehtovaara P , Söderlund H , Keränen S , Pettersson R F , Kääriäinen L
Published: May 2006

The nucleotide sequence of a nearly full-length cloned cDNA copy of an 18S defective interfering (DI) RNA of Semliki Forest virus has been determined. This corresponded to a major virus-specific cytoplasmic RNA species at the 11th undiluted passage of the virus in BHK cells. The 1652-nucleotide-long sequence consists of a unique 5'-terminal sequence followed by three tandem 484-nucleotide repeat units derived from the 5' two-thirds of the viral genome and a unique sequence of 106 nucleotides preceding the poly(A) of the 3' terminus. One of the tandem 484-nucleotide repeat units contains an extra segment of 60 nucleotides. Hybridization experiments showed that the cloned cDNA was colinear with an 18S DI RNA and that it contained an approximately 320-nucleotide-long segment colinear with the viral genomic RNA. Analysis of 18S DI RNA oligonucleotide fingerprints revealed that the molecule studied and the heterogeneous DI RNA population contain similar repeated sequences. The mechanism by which the DI RNAs are generated is not known, but it seems likely that multiple internal deletions and duplications are involved.