bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor.
Authors: Matthews B W , Ohlendorf D H , Anderson W F , Takeda Y
Published: May 2006

It is shown that the amino acid sequence and the DNA gene sequence of the 25 amino-terminal residues of the lac repressor protein of Escherichia coli are homologous with the sequences of five DNA-binding proteins: the cro repressor proteins from phage lambda and phage 434, the cI and cII proteins from phage lambda, and the repressor protein from Salmonella phage P22. The region of homology between lac repressor and the other proteins coincides with the principal DNA-binding region of cro repressor. In particular, residues Tyr-17 through Gln-26 of lac repressor correspond to the alpha-helix Gln-27 through Ala-36 of cro repressor, which we have postulated to bind within the major groove of the DNA and to be primarily responsible for the recognition of the DNA operator region by the protein [Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754--758]. By analogy with cro repressor, we propose that residues 17--26 of lac repressor are alpha-helical and that this helix and a twofold-related alpha-helix in an adjacent subunit bind within successive major grooves of the lac operator, which is in a right-handed Watson--Crick B-DNA conformation. Also, by analogy with cro repressor, we suggest that residues Thr-5 through Ala-13 of lac repressor form a second alpha-helix and contribute, in part, to DNA binding. The proposed structure for the DNA-binding region of lac repressor is consistent with chemical protection data and with genetic experiments identifying the probable locations of a number of the residues of the repressor protein that either do or do not participate in DNA binding.
The two yeast histone H2A genes encode similar protein subtypes.
Authors: Choe J , Kolodrubetz D , Grunstein M
Published: May 2006

The sequences of the two histones H2A genes in the yeast Saccharomyces cerevisiae have been determined. These genes encode two histone H2A subtypes which are 131 amino acids in length but differ at 2 amino acid positions: an Ala leads to Thr and a Thr leads to Ala change at positions 124 and 125. Thus, the two histone H2A subtypes have identical amino acid compositions. The coding regions of the two H2A genes are homologous at 369 of 393 bases (94%), with all but 2 of the 24 changes being silent. There is only 30% homology in the 5' flanking sequences of the two H2A genes. Like other eukaryotic histone genes, the yeast H2A genes are not interrupted by intervening sequences. When the yeast H2A histones are compared to those from other eukaryotes, there is at least 80% homology in amino acid sequence.
Regulation of fibrinogen synthesis by plasmin-derived fragments of fibrinogen and fibrin: an indirect feedback pathway.
Authors: Ritchie D G , Levy B A , Adams M A , Fuller G M
Published: May 2006

The effect of plasmin-derived fibrinogen fragments on the biosynthesis of fibrinogen was investigated in cultured monolayers of rat hepatocytes. Incubating the cells with several concentrations of either fibrinogen or fibrin fragment D or E had no effect on the synthesis and secretion of fibrinogen by these cells. However, if the fragments were incubated with isolated peripheral blood leukocytes, they caused these cells to secrete a factor that when added to the hepatocytes caused an increase in fibrinogen synthesis 4- to 6-fold over controls. Moreover, the hepatocyte-stimulating factor also affected the production of several other proteins produced by the hepatocyte. These results demonstrate that both fragments D and E can stimulate hepatic fibrinogen synthesis via an indirect leukocyte-mediated pathway.
cDNA clone analysis of six co-regulated mRNAs encoding skeletal muscle contractile proteins.
Authors: Hastings K E , Emerson C P
Published: May 2006

A cDNA cloning approach was used to investigate muscle gene regulation during differentiation of cultured embryonic quail myoblasts. A cDNA clone library of cultured myofiber poly(A)+RNA was constructed and screened by colony hybridization with cDNA probes of myoblast and myofiber RNA. Twenty-eight myofiber-specific cDNA clones were identified and, by cross-hybridization analysis, these clones were found to represent, at most, 18 different myofiber-specific RNAs. Six of these RNAs were identified by sequence analysis of the cDNA clones. These six RNAs encode the contractile proteins alpha-actin, alpha-tropomyosin, myosin heavy chain, myosin light chain 2, troponin C, and troponin I. The embryonic muscle contractile protein sequences are identical with, or closely match, those of adult skeletal muscle proteins and include both fast fiber (myosin light chain 2 and troponin I) and slow fiber (troponin C) isotypes. RNA gel transfer hybridization analysis showed that the cellular abundances of these contractile protein mRNAs increase 20- to 30-fold or more during myoblast differentiation. These findings indicate that coordinate activation of contractile protein synthesis during myogenesis is controlled by mechanisms that direct the accumulation of contractile protein mRNAs rather than their translational utilization. Furthermore, with the possible exception of myosin heavy chain, the contractile protein genes expressed by cultured embryogenic muscle encode adult muscle proteins of both basal and slow fiber types, consistent with a co-activation-selective repression model of gene regulation during fiber type differentiation in developing skeletal muscle.
Evidence for two dissimilar polypeptide chains in the beta 2 subunit of hexosaminidase.
Authors: Mahuran D J , Tsui F , Gravel R A , Lowden J A
Published: May 2006

The major isoenzymes of human hexosaminidase have the structures alpha beta 2 (hex A) and 2 beta 2 (hex B). In this study, we present evidence that the beta 2 subunit of hex B and hex BA (the form of hex B derived from hex A) is composed of two nonidentical polypeptide chains. We have called these chains beta a and beta b. They have similar molecular weights (25,000) but have pI values that differ by 1 unit. We have used a two-dimensional analytical gel electrophoresis method in combination with peptide mapping to compare the primary sequence structure of the two beta chains. In this method, the polypeptide chains of hex B or hex BA were first separated by isoelectric focusing in 8.5 M urea. The separated chains were subjected to partial proleolytic digestion in the stacking gel of a second NaDodSO4/polyacrylamide gel with subsequent separation of peptides by electrophoresis into the second gel. Partial digestion by protease V8 or papain showed that the beta a and beta b species have distinct primary structures, neither of which was similar to that of the alpha chain. On the basis of these results, we suggest that the beta 2 subunit of hexosaminidase has the structure of beta a beta b. The possibility that the distinct beta chains are encoded by a single gene is discussed in the light of genetic and other data.
Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.
Authors: Fulcher C A , Zimmerman T S
Published: May 2006

The human factor VIII procoagulant protein (VIIIC) was purified from the VIIIC--factor VIII--related antigen (VIIIRAg) complex in commercial factor VIII concentrate by immunoadsorbent chromatography with a monoclonal anti-VIIIRAg antibody bound to Sepharose. In this complex, VIIIC is noncovalently bound to factor VIII-related antigen. The VIIIC eluted from the complex was free of VIIIRAg as determined by immunoassay and had a specific activity of 2,294 VIIIC units/mg of protein, representing a 164,000-fold purification from plasma. Electrophoresis of this VIIIC preparation in reduced sodium dodecyl sulfate containing 5% polyacrylamide slab gels and subsequent staining with Coomassie blue showed the VIIIC to be a strongly staining doublet of Mrs 79,000 and 80,000 and more faintly staining bands of up to Mr 188,000. Treatment of the VIIIC with catalytic amounts of thrombin resulted in diminution or complete disappearance of all of these bands and appearance of a doublet of Mrs 71,000 and 72,000, a band at Mr 54,000, and material of lower Mr. The purified VIIIC was used to produce a precipitating heterologous anti-VIIIC antibody, which was shown to be monospecific for VIIIC after adsorption with factor VIII-depleted commercial concentrate. Use of this antibody in crossed immunoelectrophoresis positively identified the VIIIC bands in the sodium dodecyl sulfate/polyacrylamide gels. These techniques have allowed the identification of VIIIC protein and description of its extensive Mr heterogeneity.
Calcitonin mRNA polymorphism: peptide switching associated with alternative RNA splicing events.
Authors: Rosenfeld M G , Lin C R , Amara S G , Stolarsky L , Roos B A , Ong E S , Evans R M
Published: May 2006

Evidence is presented which supports a model proposing that differential RNA splicing events may be used in expression of genes of the endocrine system to generate alternative polypeptide hormones. We previously reported that variation in the expression of the gene encoding the small polypeptide hormone calcitonin is associated with the production of a new calcitonin-like or pseudo-calcitonin (psi Cal) mRNA. A plasmid containing psi Cal cDNA sequences has been constructed, and calcitonin genomic clones have been isolated. Hybridization analysis reveals that calcitonin and psi Cal sequences are chromosomally linked and are present in the same nuclear RNA transcripts. Both calcitonin and psi Cal mRNAs are functional and encode different polypeptide products. These data are compatible with the proposed model that alternative RNA splicing of the transcript(s) of the calcitonin gene ultimately results in the production of different polypeptide products.
Expression and novel structure of a collagen gene in Drosophila.
Authors: Monson J M , Natzle J , Friedman J , McCarthy B J
Published: May 2006

We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.
Transcription of a Drosophila heat shock gene is heat-induced in Xenopus oocytes.
Authors: Voellmy R , Rungger D
Published: May 2006

Xenopus cells, like many other eukaryotic cells, respond to heat treatments by increasing the rate of synthesis of a few characteristic proteins, the heat shock proteins. Because of the generality of this response, it seemed possible to examine the expression of isolated heat shock genes in a heterologous system. Phage 122 DNA, containing two identical genes coding for the Drosophila 70,000-dalton heat shock protein (hsp70 genes), was microinjected into Xenopus oocyte nuclei. The Drosophila hsp70 genes are transcribed efficiently in heat-treated oocytes (35-37 degrees C) to give RNA of the correct size and sequence content. Transcription is sensitive to low levels of alpha-amanitin and therefore is carried out by RNA polymerase II. At normal temperatures (20-28 degrees C) essentially no Drosophila-specific RNA is formed. The isolated insert fragment of phage 122 also gives RNA of correct length in heat-treated oocytes which hybridizes to the coding segment of Drosophila hsp70 genes only. At normal temperatures, however, its rate of transcription is variable and only RNA heterogeneous in size is formed.
Conformational dynamics of a biologically active three-fragment complex of horse cytochrome c.
Authors: Juillerat M , Taniuchi H
Published: May 2006

The conformational dynamics of a biologically active noncovalent complex containing three fragments, ferroheme fragment (1-25)H and apofragments (28-38) and [3H](56-104) [or [3H](39-104)], of horse cytochrome c has been studied with respect to kinetics and thermodynamics of dissociation. The rate of unfolding of the two-fragment complex ferro(1-25)H . (56-104) was also estimated. The results indicate that the ferrous three-fragment complex exhibits a higher frequency of dissociation-association with fragment (28-38) and a lower frequency of overall unfolding-folding at pH 7.0. In the presence of an excess of free (28-38) and below 30 degrees C, unfolding of the ferrous three-fragment complex appears to occur by activation to the transitional state without a large change in conformation, followed by virtually simultaneous dissociation of all three of the fragments [without going through the complex (1-25)H . (56-104), which is a major intermediate for folding]. Above 30 degrees C unfolding via the complex (1-25)H . (56-104) becomes detectable because the equilibrium between the two- and the three-fragment complex is highly temperature dependent. Thus, the relative probabilities of these two different ways of transition for unfolding are modulated by temperature. The observations suggest that the mode of activation of protein and hence the pathway for unfolding may vary depending on temperature. It is also suggested that the interatomic interactions binding the three fragments together in the ordered complex are linked to strengthen each other in the ground state.
Genetic analysis of the role of cAMP in mediating effects of interferon.
Authors: Schneck J , Rager-Zisman B , Rosen O M , Bloom B R
Published: May 2006

The effects of interferon (IFN) on Fc receptor-mediated phagocytosis, intracellular cAMP levels, antiviral activity, and growth inhibition were analyzed in a cloned macrophage-like cell line, J774.2, and variants derived from it. Purified IFN increased Fc receptor-mediated phagocytosis in J774.2 cells, and in cAMP-responsive nonphagocytic variants but was without effect in cAMP-unresponsive nonphagocytic variants, in adenylate cyclase-deficient variants, and in cAMP-dependent protein kinase-deficient variants. Under conditions in which IFN augmented phagocytosis, it increased intracellular levels of cAMP. Parental cells were highly sensitive to IFN-mediated growth inhibition. In contrast, cAMP-dependent protein kinase-deficient variants were only 1/100th as sensitive to growth inhibition by IFN. All cell lines tested, both responsive and unresponsive to cAMP, were equally protected by IFN against infection with vesicular stomatitis virus, demonstrating that the antiviral state was independent of cAMP. These results indicate that, in transformed macrophages, stimulation of phagocytosis and inhibition of growth by IFN are mediated through intracellular cAMP, whereas the antiviral state induced by IFN is independent of cAMP.
Cell surface insertion of exogenous epidermal growth factor receptors into receptor- mutant cells: demonstration of insertion in the absence of added fusogenic agents.
Authors: Bishayee S , Feinman J , Pittenger M , Michael H , Das M
Published: May 2006

We show that epidermal growth factor (EGF) receptor can be transferred in a biologically active orientation from donor hepatic membranes to recipient receptorless fibroblast cells. The recipient cells (NR-6) normally lack EGF receptors and are biologically unresponsive to EGF. The transfer of receptors from donor plasma membranes to recipient NR-6 surface membranes occurs in the absence of any added fusogenic agent. Studies on time and temperature dependence of this transfer indicate that it is due to preferential insertion of the EGF receptor over the other hepatic proteins. The inserted receptor is exceptionally stable to dissociation or damage, and this facilitated studies on its biological properties. The inserted receptor confers upon the hitherto unresponsive variant NR-6 cells a specific biological responsiveness to EGF as measured by EGF-induced stimulation of DNA replication and cell division. These findings suggest the existence of an affinity-mediated mechanism for the biologically active insertion of exogenous EGF receptors into receptorless variant cells. This insertion approach may be of use in the identification of receptor-associated membrane proteins that play a role in the transmission of EGF biological message.
Full-term development after transplantation of parthenogenetic embryonic nuclei into fertilized mouse eggs.
Authors: Hoppe P C , Illmensee K
Published: May 2006

Diploid parthenogenetically activated oocytes were obtained after gonadotropin-induced ovulation of virgin females of the LT/Sv (LT) inbred mouse strain. These oocytes cleave spontaneously and develop into blastocysts which implant in the uterus but die within a few days. We examined the developmental potential of nuclei from parthenogenetic embryos after transplantation into fertilized eggs. The inner cell mass (ICM) and trophectoderm (TE) of LT parthenogenetic blastocysts were mechanically isolated and dissociated into single cells. Their nuclei were then injected into fertilized C57BL/6J eggs from which the male and female pronuclei were removed. Of 94 eggs injected with TE cell nuclei, 4 embryos developed to the morula stage; all 4 showed abnormalities and subsequently became arrested in development. Enzyme analysis of these embryos revealed that TE cell nuclei could neither independently initiate or support preimplantation development. However, of 54 eggs injected with nuclei from ICM cells, 3 morulae and 3 blastocysts developed and enzyme analyses of them confirmed that the preimplantation development of 2 embryos was supported by transplanted parthenogenetic nuclei. In another experimental series, 3 morulae and 4 blastocysts developed from 107 eggs injected with ICM nuclei and were transferred to uteri of foster mothers to ascertain their postimplantation development. Four female offspring were born and all of them showed a diploid karyotype and expressed enzyme activity of only the LT genotype. One female proved to be fertile and transmitted the parthenogenetic genome to the next generation. These results demonstrate that the nucleus from LT parthenogenetic blastocysts contains a complete genome necessary to support development of an adult mouse. Therefore, the early postimplantation death of parthenogenetic embryos does not seem to be related to an aberrant genotype but rather to undefined mechanisms associated with fertilization and normal morphogenetic processes.
Complementation of the defects of DNA synthesis in irradiated and unirradiated ataxia-telangiectasia cells.
Authors: Murnane J P , Painter R B
Published: May 2006

Mutant subtypes in the human genetic disease ataxia-telangiectasia (A-T) were classified by means of an assay system that monitors complementation of defects in DNA synthesis. Anomalies in DNA synthesis have been observed previously in A-T cells, both in their failure to inhibit DNA synthesis immediately after exposure to ionizing radiation and in their prolonged S phase. Polyethylene glycol-mediated cell fusion and autoradiography were combined with selective identification of different A-T cell populations by fluorescent colored microspheres to determine complementation capabilities of various A-T cell combinations. Five complementation groups were identified by both a 30-40% increase in the rate of DNA synthesis in unirradiated heterokaryons and the appearance of normal inhibition of DNA synthesis after x-irradiation of heterokaryons. The correlation observed between these phenomena suggests that the defects in A-T cells involve problems in initiation of DNA synthesis.
Dissociation of two signals required for activation of resting B cells.
Authors: Julius M H , von Boehmer H , Sidman C L
Published: May 2006

Cellular interactions involved in the T cell-dependent activation of B cells were analyzed by using lines and clones of helper T cells specific for determinants expressed on the B cell surface. Activation of male antigen-, M locus-, and H-2-specific T cells was shown to support polyclonal Ig production by a population of B cells that did not require T-cell-B-cell interaction for induction/amplification. However, these T cells alone did not activate gradient-purified small (resting) B cells. The activation of small B cells was shown to require not only a signal derived through an antigen-specific T-helper cell-B cell interaction but in addition a second signal that could be provided by anti-Ig antibodies.
Molecular forms of the putative enkephalin precursor BAM-12P in bovine adrenal, pituitary, and hypothalamus.
Authors: Baird A , Ling N , Böhlen P , Benoit R , Klepper R , Guillemin R
Published: May 2006

A highly specific radioimmunoassay for one of the putative adrenomedullary [Met]enkephalin precursors, BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-OH), has been developed. The BAM-12P antibodies recognize the COOH-terminal fragment of the peptide from Arg7 to Glu12 and do not crossreact with [Met5]- or [Leu5]enkephalin or any of their COOH-terminal lysine or arginine extended analogs. Specificity for the COOH-terminal Glu-OH is suggested by the 100% crossreactivity with BAM-12P5-12 and 0.3% crossreactivity with BAM-12P5-12 amide. Using these antibodies, we have measured three forms of BAM-12P-like immunoreactivity in extracts of bovine adrenal medulla, of which the major form (greater than 90%) corresponds to BAM-12P by molecular weight. Extracts of bovine adrenal cortex contain 1% the amount of a BAM-12P-like material (Mr approximately 1400; 20 ng per gland), possibly due to crosscontamination with adrenomedullary tissue. The major form of BAM-12P-like material in extracts of bovine neurointermediate pituitaries is of higher molecular weight than authentic BAM-12P (Mr approximately 4000); the remaining material (10%) corresponds to BAM-12P by molecular weight. There is no detectable BAM-12P-like immunoreactivity in crude or purified extracts of bovine anterior pituitaries. Extracts of bovine hypothalamic tissues contain small amounts of BAM-12P immunoreactivity (approximately 2 ng per fragment) which can be detected as one molecular form corresponding to a 1400-dalton molecule. The results indicate that the enkephalin precursor found in the adrenal medulla also may be present in the pituitary and hypothalamus. Furthermore, the processing of this molecule appears to be tissue-specific.
Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity.
Authors: Tokes Z A , Rogers K E , Rembaum A
Published: May 2006

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not increase their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.
Inhibition of amiloride-sensitive sodium conductance by indoleamines.
Authors: Legris G J , Will P C , Hopfer U
Published: May 2006

To examine a possible role of indoleamines in the regulation of epithelial sodium absorption, the effect of serotonin (5-hydroxytryptamine) and several derivatives on electrolyte transport was measured in vitro in the baboon bronchus and in the trachea and colon of sodium-deficient rats. Serotonin, melatonin (N-acetyl-5-hydroxytryptamine), and harmaline (1-methyl-7-methoxy-3,4-dihydro-beta-carboline) inhibited sodium transport in all three preparations in a similar manner to the natriuretic agent amiloride. In all three epithelia, sodium absorption via the amiloride-sensitive pathway constitutes a substantial portion of total electrolyte transport, measured as the amiloride-sensitive short-circuit current. Thus 25 microM amiloride inhibited the short-circuit current 21% in the rat trachea, 63% in the baboon bronchus, and 90% in the rat colon. Serotonin, melatonin, and harmaline inhibited the amiloride-sensitive portion of the short-circuit current from the luminal side of the epithelium. The inhibition was rapid, requiring only seconds, and maximal inhibition by serotonin was identical to that by amiloride. When sodium was omitted from the luminal solution, the short-circuit current was reduced a similar amount, suggesting that sodium absorption was being inhibited by both amiloride and the indoles. The IC50 value for amiloride was 50 nM in the baboon bronchus and 500 nM in the rat colon. In contrast, the IC50 value for serotonin was 0.4 mM in the baboon bronchus and 8 mM in the rat colon. These results, together with the wide distribution of amine-precursor-uptake-and-decarboxylation (APUD) cells in the respiratory and intestinal tract, suggest that certain indoleamines could play a role as local regulators of fluid and electrolyte transport. For example, in the airways, indoleamines may be one of the factors involved in regulation of the depth of the periciliary fluid layer.
Renal transport of taurine adapts to perturbed taurine homeostasis.
Authors: Rozen R , Scriver C R
Published: May 2006

Renal adaptation apparently contributes to the homeostasis of taurine, a beta-amino compound that behaves as a conserved metabolite in the mammal. We studied two strains of inbred mice: C3H/HeJ (low-taurine excreter) and C57BL/6J (high-taurine excreter due to impaired basolateral membrane permeability to taurine). Low-protein and low-sulfur amino acid diets fed for two weeks significantly decreased plasma taurine in both strains, decreased fractional taurine excretion in vivo (particularly in the C57BL strain), and increased net uptake of taurine by renal cortex slices and isolated brush-border membrane vesicles (BBMV) in vitro in both strains. Renal adaptation was less obvious in vivo in the low-taurine excreter C3H strain, but in vitro adaptation, as observed in slices and BBMV (P less than 0.01), was greater than that observed in the C57BL strain. Renal cellular taurine content fell (P less than 0.01) only in the adapted C3H strain. The in vitro adaptive response was not confined to taurine; BBMV uptake of D-glucose and L-alanine was also enhanced in the adapted state. Specificity of the stimulus for adaptation was tested with a low-phenylalanine diet; a modest adaptation was observed in vivo and in vitro but only in the C3H strain. BBMV adaptation did not correlate with blood methionine but correlated inversely with plasma taurine (r = 0.71, P less than 0.05), implying that change in extracellular taurine may be a signal for renal adaptation in taurine homeostasis in the mammal.
Discoidins I and II: common and unique regions on two lectins implicated in cell--cell cohesion in Dictyostelium discoideum.
Authors: Berger E A , Armant D R
Published: May 2006

As Dictyostelium discoideum amoebae differentiate from the noncohesive to the mutually cohesive state, they synthesize two galactose-binding lectins--discoidins I and II--which have been implicated as obligatory components of the morphogenetic cell-cell recognition and cohesion system. These proteins have been shown to have similar amino acid compositions and subunit Mr and overlapping but distinct carbohydrate recognition specificities. We have performed extensive immunochemical and biochemical analyses to study the structural relationships between these two molecules and to eventually identify structural and functional domains. Antisera raised against highly purified preparations of discoidin I and discoidin II were tested for their reactivities against each protein by both immunoprecipitation and double diffusion analyses. The patterns of crossreactivity indicated the presence of shared as well as unique antigenic determinants. This interpretation was supported by two-dimensional thin-layer peptide map analysis and by studies with purified peptides. Of approximately 10-12 peptides observed after exhaustive tryptic digestion of each radioiodinated lectin, 3 appeared to be common to both. These putative common peptides were purified, and the corresponding peptides from discoidins I and II were found to behave identically by two-dimensional thin-layer analysis, gel filtration, and susceptibility to chymotrypsin. The finding of common and unique regions in discoidins I and II suggests analogies with other families of recognition proteins and may have important functional implications for these cell-cell recognition molecules.
Methylation of the viral genome in an in vitro model of herpes simplex virus latency.
Authors: Youssoufian H , Hammer S M , Hirsch M S , Mulder C
Published: May 2006

An in vitro model of latency of herpes simplex virus type 1 (HSV-1) in a lymphoid cell line has been developed recently. CEM cells persistently infected with HSV-1 transiently ceased to produce virus for 24 days. This nonproductive state could either be reversed with phytohemagglutinin or maintained with concanavalin A. This system was used to study the relationship between DNA methylation and HSV-1 latency. DNA was probed for methylation by comparing the cleavage patterns generated by two pairs of restriction endonucleases (Sma I vs. Xma I and Hpa II vs. MspI); these enzymes show differential activity reflecting methylation of the recognition sequences. Viral DNA in the concanavalin A-treated cells (not producing virus) was found to be extensively methylated. By contrast, no methylated copies were detected in viral DNA from producer cells. About 800 days after the initial infection, the productive culture once again became nonproductive. Viral sequences in the latter cells were also methylated. Reconstitution experiments revealed 1-2 copies of viral DNA in cells from the latent stages and 40-80 copies in cells from productive stages. Most (if not all) of the viral genome is present in cells from various productive and latent stages. No differences in sequence arrangement were detected (although a terminal fragment of intracellular HSV-1 DNA appeared to be under-represented in latent cells). These results suggest a role for DNA methylation in the mechanism of HSV-1 latency in this system.
Function of adenovirus terminal protein in the initiation of DNA replication.
Authors: Tamanoi F , Stillman B W
Published: May 2006

An early event in the initiation of adenovirus DNA replication is the formation of a covalent complex between the 87,000-dalton adenovirus terminal protein precursor and 5'- dCMP (pTP-dCMP complex). Nuclear extracts prepared from adenovirus-infected HeLa cells catalyzed complex formation in the presence of ATP, Mg2+, and adenovirus DNA-protein complex but were not active when Pronase-treated DNA was used as template. The activity has been partially purified by chromatography on denatured DNA-cellulose and used to examine whether the 55,000-dalton terminal protein on adenovirus DNA is required for pTP-dCMP complex formation. Results obtained with either DNA-protein complex or Pronase-treated DNA were identical to those obtained using crude nuclear extracts. However, after treatment with piperidine to remove residual peptides. Pronase-treated DNA supported complex formation with the partially purified activity but not with the crude extracts. In addition, when a plasmid containing an origin of adenovirus DNA replication was used as template, the pTP-dCMP complex was formed provided the plasmid was linearized in such a way that the origin was located at the end of the molecule. Neither linearized plasmid DNA with an internal origin nor supercoiled plasmid DNA supported complex formation. Furthermore, after heat denaturation, the linear plasmid DNA still supported complex formation, again provided that the origin was located at the end of the molecule. The partially purified protein fraction supported a limited amount of DNA chain elongation, which permitted exact positioning of the initiation site. These results suggest that enzymes responsible for complex formation recognize a DNA sequence at the origin and that the terminal protein on the template DNA plays a subordinate role.
Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide.
Authors: Mollay C , Vilas U , Kreil G
Published: May 2006

It has previously been shown that rat liver microsomes contain a proteolytic enzyme that cleaves honeybee prepromelittin to yield promelittin. This enzyme has now been further purified by centrifugation on a sucrose-deoxycholate gradient and then reconstituted into phospholipid vesicles. Incubation of prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with l-butanol and paper electrophoresis in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. The microsomal enzyme is thus an endoprotease that hydrolyzes prepromelittin exclusively at the pre-pro junction. The precision of this cleavage of an insect preprotein by a rat liver enzyme indicates that we are dealing with the ubiquitous eukaryotic signal peptidase.
Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.
Authors: Watson J C , Surzycki S J
Published: May 2006

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.
Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation.
Authors: Doehmer J , Barinaga M , Vale W , Rosenfeld M G , Verma I M , Evans R M
Published: May 2006

We have introduced the rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector. The ability of the viral DNA to induce foci in the recipient cells was used as a dominant selection marker. Several copies of rat growth hormone DNA were integrated in the mouse cells. The transformed mouse cells expressed rat growth hormone-specific mRNA and secreted mature rat growth hormone. In rat cells, the expression of this gene is regulated by glucocorticoids. We demonstrate that hormone-dependent regulation transfers with the clone and thus appears to be an intrinsic property of the gene or its RNA products.
Paradoxical effects of glucocorticoids on regulation of plasminogen activator activity of rat hepatoma cells.
Authors: Barouski-Miller P A , Gelehrter T D
Published: May 2006

Incubation of rat hepatoma cells with cAMP derivatives stimulates cell-associated plasminogen activator activity 8- to 22-fold and extracellular plasminogen activator activity 30- to 1300-fold. This time- and concentration-dependent increase is enhanced by phosphodiesterase inhibitors. Dexamethasone, a synthetic glucocorticoid, decreases the plasminogen activator activity of these cells, probably through induction of an inhibitor. Paradoxically, dexamethasone, added simultaneously with cAMP derivatives causes a further 4-fold enhancement of the cAMP-mediated stimulation of plasminogen activator activity. Dexamethasone also alters the time course of cAMP-mediated enhancement of plasminogen activator activity: increased protease activity is detected at 4 hr in cells incubated with 8-bromoadenosine-3':5'-cyclic monophosphoric acid and 1-methyl-3-isobutylxanthine but not until 12 hr in cells incubated with dexamethasone as well. Glucocorticoids thus exert two separate and opposite effects on plasminogen activator activity: induction of an inhibitor and amplification of cyclic nucleotide action. Although permissive and synergistic effects of dexamethasone on cyclic nucleotide action have been reported previously, glucocorticoid regulation of plasminogen activator activity is unique in that the amplification of cyclic nucleotide effects by dexamethasone opposes its regulatory action toward a specific enzyme.
Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin.
Authors: Ingolia T D , Craig E A
Published: May 2006

The primary base sequence of the protein coding regions of the four small heat shock genes of Drosophila melanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show striking homologies over approximately 50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of approximately 200 amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian alpha-crystallin. The possible functional significance of this structural similarity is discussed.
Enrichment of human marrow lymphocytes with monoclonal antibodies to murine antigens.
Authors: Landreth K S , Kincade P W , Lee G , Gathings W E , Fu S M
Published: May 2006

Two monoclonal antibodies, prepared against a murine B lymphoma and characterized as binding to a cell surface antigen represented primarily on cells of the B lineage, were found to bind to human hemopoietic cells. These antibodies recognize similar populations of cells in mice and humans. Antibodies from clones 177.17 and 83.4 bound to 6% of human bone marrow nucleated cells. This included all cells with detectable cell surface Ig (sIg+) and those that lack sIg but have detectable cytoplasmic mu (sIg-, c mu+), considered to be the immediate precursors of B lymphocytes (pre-B cells). In addition, these antibodies bound to a subpopulation of T cells and a proportion of null lymphocytes in marrow, spleen, and peripheral blood. An unexpected finding was that established pre-B cell lines were not recognized by these antibodies and possible reasons for this are considered. By using antibody-coated polystyrene plates for cell depletion and recovery, highly enriched preparations of c mu+, sIg- cells have been obtained. These antibodies and enrichment procedures should prove valuable in establishing the minimal requirements for maturation of these putative precursors in vitro, for comparative studies of immunodeficiency/autoimmune diseases in man and experimental animal models, and for monitoring the outcome of therapeutic marrow transplantation.
Construction of DNA recognition sites active in Haemophilus transformation.
Authors: Danner D B , Smith H O , Narang S A
Published: May 2006

Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer.
Junction between Z and B conformations in a DNA restriction fragment: evaluation by Raman spectroscopy.
Authors: Wartell R M , Klysik J , Hillen W , Wells R D
Published: May 2006

Raman vibrational spectra were obtained from two DNA restriction fragments and the DNA polymer (dG-dC)n . (dG-dC)n in 0.01 and 4.5 M NaCl. One fragment contained 95 base pairs (bp) of the Escherichia coli lactose operator-promoter region (95-bp fragment). The other fragment consisted of the 95-bp region flanked by 26 and 32 bp of dC-dG sequences and BamHI ends (157-bp fragment). In 0.01 M NaCl all three DNAs have Raman spectra characteristic of a right-handed B conformation. The high salt spectrum of the 95-bp fragment is also characteristic of a B conformation. However, the spectrum of the 157-bp fragment in 4.5 M NaCl shows major intensity changes from the 0.01 M NaCl spectrum. These changes are also observed in the high salt spectra of (dG-dC)n . (dG-dC)n and are correlated with the presence of a left-handed Z conformation. Comparisons between the high salt Raman spectra of the 157-bp fragment and spectra calculated from (dG-dC)n . (dG-dC)n and the 95-bp fragment indicated that essentially all of the dC-dG regions in the 157-bp fragment are in the Z conformation and a large part (approximately 80%) of the 95-bp region no longer has a B-type backbone vibration. However, this non-B-DNA-like character of the central region is not indicated by base vibrations.
Neural networks and physical systems with emergent collective computational abilities.
Authors: Hopfield J J
Published: May 2006

Computational properties of use of biological organisms or to the construction of computers can emerge as collective properties of systems having a large number of simple equivalent components (or neurons). The physical meaning of content-addressable memory is described by an appropriate phase space flow of the state of a system. A model of such a system is given, based on aspects of neurobiology but readily adapted to integrated circuits. The collective properties of this model produce a content-addressable memory which correctly yields an entire memory from any subpart of sufficient size. The algorithm for the time evolution of the state of the system is based on asynchronous parallel processing. Additional emergent collective properties include some capacity for generalization, familiarity recognition, categorization, error correction, and time sequence retention. The collective properties are only weakly sensitive to details of the modeling or the failure of individual devices.
Prediction of the shapes of deformed nuclei by the polyspheron theory
Authors: Pauling Linus
Published: May 2006

It is pointed out that some features of the shapes of
62
154
Sm,
68
166
Er, and
70
176
Yb as reported by Cooper
et al.
[Cooper, T., Bertozzi, W., Heisenberg, J., Kowalski, S., Turchinetz, W., Williamson, C., Cardman, L., Fivozinsky, S., Lightbody, J., Jr., & Penner, S. (1976)
Phys. Rev. C
13, 1083-1094] from electron-scattering experiments agree reasonably well with the description of these nuclei, derived from the values of the rotational energy levels, as consisting of a large spherical part (essentially
132
Sn) and a cap [Pauling, L. (1969)
Proc. Natl. Acad. Sci. USA
64, 807-809; Pauling, L. & Robinson, A. B. (1975)
Can. J. Phys.
53, 1953-1964].
Mutagenicity of oxygen free radicals.
Authors: Moody C S , Hassan H M
Published: May 2006

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) was used as an intracellular generator of oxygen free radicals and was found to be highly mutagenic for Salmonella typhimurium. It caused both base-pair substitution and frameshift mutations. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium. The mutagenicity of paraquat was dependent upon the presence of a supply of both electrons and oxygen. Cells containing high levels of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) were more resistant to the toxicity and the mutagenicity of paraquat than were cells containing normal levels of this enzyme. The mutagenicity of paraquat thus appears to be due to its ability to exacerbate the intracellular production of superoxide radicals.
Activation of purified soluble guanylate cyclase by protoporphyrin IX.
Authors: Ignarro L J , Wood K S , Wolin M S
Published: May 2006

Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] purified from bovine lung is markedly activated (30- to 40-fold) by protoporphyrin IX (Ka, 15-25 nM) and is inhibited by hematin (Ki, 3.7 microM) when MgGTP is used as substrate. Guanylate cyclase possesses specific activities (mumol of cGMP per min/mg of protein) of 0.1-0.2 (MgGTP) and 0.3-0.5 (MnGTP) and can attain values of 2-8 (MgGTP) or 1-1.4 (MnGTP) in the presence of protoporphyrin IX. Guanylate cyclase purified in this study contains heme and is activated by nitric oxide and nitrosyl-heme to the same magnitude as that by protoporphyrin IX. With the exception of hematoporphyrin IX, close structural analogs of protoporphyrin IX, including precursors and metabolites, do not activate guanylate cyclase. The insertion of iron into protoporphyrin IX to form heme or hematin renders the metalloporphyrin an inhibitor of unactivated or activated guanylate cyclase. The data suggest that protoporphyrin IX and heme could function to modulate guanylate cyclase activity.