bluuebunny/crossref_metadata_2025 huggingface dataset:
A model for catabolite activator protein binding to supercoiled DNA
Authors: Salemme F. R.
Published: May 2006

Catabolite activator protein (CAP) is a dimeric molecule (
M
r
= 2 × 22,500) involved in transcription initiation of several catabolite-sensitive genes of
Escherichia coli
. The present communication proposes a model for the interaction of CAP with DNA. The model is based upon known geometrical features of the CAP molecule [McKay, D. B. & Steitz, T. A. (1981)
Nature
(
London
) 290, 744-749], which allow interaction between dyad-related α-helices of the dimeric protein and major grooves in adjacently aligned sections of right-handed B-DNA. These geometrical features suggest that
in vivo
CAP binding to closed-circular DNA involves CAP bridging adjacent loops of a DNA solenoidal coil. This interaction pattern is shown to be consistent with the geometrical and stoichiometric properties of nonspecifically bound CAP complexes observed by Chang
et al.
[Chang, J. J., Dubochet, J., Baudras, A., Blazy, B. & Takahashi, M. (1981)
J. Mol. Biol.
150, 435-439]. CAP-induced coil formation is related to
in vivo
CAP potentiation of RNA polymerase activity in underwound closedcircular DNA. Specifically, it is proposed that CAP binding to the right-interwound form of supercoiled DNA effects a local redistribution of DNA twist-strain energy, thus resulting in the formation of a left-handed solenoidal loop. The production of this localized solenoidal loop, which reflects compensatory alterations in DNA twist and writhe, may provide a conformationally unique site for RNA polymerase binding where the DNA is partially unwound. The proposed interaction pattern is consistent with both recent DNA unwinding experiments and various nuclease protection data. Moreover, features of the model suggest that the repetitive and symmetric character of many promoter sequences may provide the structural basis for a switching mechanism operative in the differential control of gene transcription.
DNA methylation in chicken alpha-globin gene expression.
Authors: Haigh L S , Owens B B , Hellewell O S , Ingram V M
Published: May 2006

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.
Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.
Authors: Pandey K N , Sabharwal P S
Published: May 2006

Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through in vitro activation of hormone synthesis in callus cells.
Microinjected progesterone reinitiates meiotic maturation of Xenopus laevis oocytes.
Authors: Tso J , Thibier C , Mulner O , Ozon R
Published: May 2006

Microinjection of progesterone dissolved in paraffin oil induces the reinitiation of meiotic maturation in the Xenopus oocyte; 50% maturation is obtained when 50 nl of a 50 microM solution is microinjected into the oocyte. The kinetics of the response to microinjected progesterone are similar to the kinetics of response to externally applied hormone. When an aqueous solution of progesterone is microinjected instead of an oil solution, maturation is never observed, a result which confirms previous work. Leakage of the steroid into the external medium was estimated to range from 1.6 pmol/hr when microinjection was performed in oil to 3.6 pmol/hr when it was performed in aqueous solution. Metabolism of the hormone microinjected in oil is weak (less than 20%) as compared to that after aqueous microinjection (greater than 80%). Progesterone microinjected in oil decreases the cAMP content as does externally applied hormone. We therefore conclude that progesterone acts initially on an intracellular site in order to trigger meiotic maturation of the Xenopus oocyte.
Cellular analysis of specificity of antibodies and of delayed type hypersensitivity responses toward some structurally related synthetic antigens: Boosting is determined by specificity of T cells
Authors: Parhami-Seren Behnaz , Strassmann Gideon , Mozes Edna , Sela Michael
Published: May 2006

The crossreactivity between the random synthetic polypeptide antigen poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] and its ordered-sequence analogs (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys) [(T-T-G-G)-A--L] and (Tyr-Glu-Tyr-Glu)-poly(DLAla)--poly(Lys) [(T-G-T-G)-A--L] at the level of humoral and cellular responses was studied. For delayed type hypersensitivity responses, (T,G)-A--L-activated T cells could be challenged with the homologous antigen as well as with the ordered analogs. T cells activated by (T-T-G-G)-A--L could be challenged with either the homologous antigen or with (T,G)-A--L but not with (T-G-T-G)-A--L. Similarly, no cross stimulation was observed between (T-G-T-G)-A--L-activated cells and (T-T-G-G)-A--L, whereas (T,G)-A--L could challenge the latter cells to mediate significant responses. Similar but not identical cross reactions were observed when primed spleen cells or lymph nodes were transferred to irradiated recipients that were boosted for the production of antibodies. In contrast to observations at the level of cellular responses, (T-G-T-G)-A--L-primed spleen or lymph node cells could not be boosted with (T,G)-A--L for the production of detectable amounts of antibodies, although boosting with the homologous antigen resulted in significant levels of (T-G-T-G)-A--L-specific antibodies. Transfer experiments in which mixtures of T and B cells, each primed to a different ordered polypeptide antigen, were injected into irradiated recipients showed that successful cooperation occurs provided that the boost is given with the T-cell-specific antigen. The antibodies produced were specific to the antigen used for B-cell priming. The T-cell-B-cell collaboration probably occurs through specific determinants that are shared between the two antigens in which the ordered peptides are attached to the same multichain polymer and that are recognized by both the T and the B cells.
T-lymphocyte heterogeneity: wheat germ agglutinin labeling of transmembrane glycoproteins.
Authors: Torrisi M R , Da Silva P P
Published: May 2006

We have recently described "fracture-label" techniques that permit direct cytochemical labeling of freeze-fractured cells. We report here the use of fracture-labeling to investigate the distribution and partition of wheat germ agglutinin (WGA) receptor sites over the protoplasmic and exoplasmic plasma membrane faces of freeze-fractured human thymus-derived (T) lymphocytes. All exoplasmic faces are strongly labeled by WGA. In contrast, the protoplasmic faces exhibit remarkable variation, ranging from virtual absence of label in some faces to very high densities in other faces. We interpret the presence of WGA receptor sites over the protoplasmic faces to reflect the presence of transmembrane WGA-binding sialoglycoproteins that, during freeze-fracture, partition with the inner half of the plasma membrane. Our results, therefore, indicate heterogeneous expression of integral membrane proteins within populations of human T cells. Fracture-label techniques thus represent an additional tool in the definition of lymphocyte subpopulations.
Kappa opiate receptors localized by autoradiography to deep layers of cerebral cortex: relation to sedative effects.
Authors: Goodman R R , Snyder S H
Published: May 2006

Kappa opiate drugs differ from other opiates in their unique sedative actions and lack of cross-tolerance. We have visualized kappa opiate receptors by in vitro autoradiography using the kappa drugs [3H]ethylketazocine ([3H]EKC) and [3H]bremazocine. Though these ligands also label mu and delta opiate receptors, their binding is rendered kappa specific by coincubation with morphine and [D-Ala2, D-Leu5]enkephalin (DADL-Enk) to displace mu and delta interactions, respectively. Labeling patterns with [3H]EKC and [3H]bremazocine are the same and differ markedly from localizations of mu and delta opiate receptors visualized with [3H]dihydromorphine and [3H]DADL-Enk, respectively. The highest density and most selective localization of putative kappa receptors occurs in layers V and VI of the cerebral cortex. In these layers cells are localized which project to the thalamus regulating sensory input to the cortex. Receptors in these layers could account for the unique sedative and possibly analgesic effects of kappa opiates.
A genetic locus regulates the expression of tissue-specific mRNAs from multiple transcription units.
Authors: Levy D E , Lerner R A , Wilson M C
Published: May 2006

129 GIX- mice, unlike animals of the congeneic partner strain GIX+, do not express significant amounts of the retroviral antigens gp70 and p30. Evidence is presented indicating that the GIX phenotype is specified by a distinct regulatory gene acting on multiple transcription units to control the levels of accumulation of specific mRNA species. The steady-state levels of retroviral-homologous mRNA from the tissues of GIX+ and GIX- mice were examined by blot hybridization using as probes DNA fragments from cloned murine leukemia viruses. RNA potentially encoding viral antigens was reduced or absent in GIX- mice, even though no differences in integrated viral genomes were detected between these congeneic strains by DNA blotting. Tissue-specific patterns of accumulation of these RNA species were detected in brain, epididymis, liver, spleen, and thymus, and several distinct RNA species were found to be coordinately regulated with the GIX phenotype. Measurements of RNA synthesis suggest a major role for transcriptional control in the regulation of some retroviral messages.
Mechanism of tobacco mosaic virus assembly: role of subunit and larger aggregate protein.
Authors: Fukuda M , Okada Y
Published: May 2006

Tobacco mosaic virus (TMV) was reconstituted from the RNA of a common strain (OM) and the protein of a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is a member of the tobamovirus group. In 0.25 M phosphate buffer at 25 degrees C, CGMMV-W protein existed mainly as 21S aggregates. When this protein was mixed with OM RNA, complexes of short rods were formed but further elongation did not occur. After the addition of subunits in 0.1 M phosphate buffer at 25 degrees C, elongation to the 5' end of the RNA proceeded as fast as in the case of reconstitution with the usual equilibrium "disk preparation" of OM protein, to give 260-nm intermediates in the first 5-7 min. The results proved that the rapid elongation we previously observed in the reconstitution of TMV-OM following the assembly initiation is the outcome of preferential incorporation of TMV subunit protein. Either preformed 21S aggregate or the subunit of CGMMV protein was added to the 260-nm intermediate. Elongation to the 3' end of the RNA was investigated in 0.1 M phosphate buffer at 25 degrees C by measuring the distribution of rod length and the RNase-resistant infectivity. The results showed that the 21S aggregate is kinetically favored as the protein source during the slow elongation process.
Translational block to expression of the Escherichia coli Tn9-derived chloramphenicol-resistance gene in Bacillus subtilis.
Authors: Goldfarb D S , Rodriguez R L , Doi R H
Published: May 2006

The Gram-negative product-encoding Tn9-derived chloramphenicol-resistance (Cmr) gene can be cloned but not phenotypically expressed in Bacillus subtilis. We show that, even when transcribed from B. subtilis promoters, the ribosomal binding site for the Cmr gene does not function well in B. subtilis. The Cmr gene product, chloramphenicol acetyltransferase (CmAcTase; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), is detected in B. subtilis when the promoters, ribosomal binding sites, and initiation codons of B. subtilis genes are fused to the Cmr gene. These gene fusions lead to the in vivo production of mRNAs containing B. subtilis translation start signals followed in an open reading frame by the translation start site normally used by Escherichia coli to initiate translation of Cmr mRNA. Both fusion and native CmAcTase proteins are produced in E. coli, but only fusion CmAcTase is produced in B. subtilis. We conclude that the absence of native CmAcTase in B. subtilis is due to inability of the E. coli ribosomal binding site to function well in B. subtilis. Since fusion CmAcTase polypeptides are produced in E. coli, we conclude that these particular B. subtilis regulatory elements function heterologously in E. coli. The absence of a suitable binding site on the Cmr gene for B. subtilis ribosomes is consistent with reports that many E. coli genes are not expressed in B. subtilis and that E. coli mRNA functions poorly in B. subtilis in vitro translation systems. The functioning of B. subtilis regulatory sequences in E. coli is consistent with in vivo and in vitro data showing the expression of B. subtilis genes in E. coli. To confirm the hypothesis that the large CmAcTase proteins are NH2-terminal fusions of native CmAcTase we partially determined the sequence of one CmAcTase fusion protein.
Mapping the mitochondrial DNA of
Zea mays
: Ribosomal gene localization
Authors: Iams Keith P. , Sinclair John H.
Published: May 2006

We have located the 18S and 26S ribosomal genes on a 32.2-kilobase pair (kb) restriction map of
Zea mays
mitochondrial DNA. In a
Bam
HI restriction digest of mitochondrial DNA, band 4 carries all of the 26S gene whereas band 2 carries the 18S gene sequence. We have cloned and mapped bands 2 and 4 and show that they are contiguous in the genome. The 26S sequence is at one end of the 13.7-kb fragment 4, immediately adjacent to the junction with fragment 2. The 18S sequence is located at the far end of the 17.5-kb fragment 2, about 15 kb away from the 26S gene. A second region of 18S sequence homology is found on band 40. This region contains sequences that cross-hybridize with those in band 2. The nature of this apparent sequence repetition is unclear.
Purification of a prolactin receptor.
Authors: Liscia D S , Vonderhaar B K
Published: May 2006

A prolactin receptor was purified from a solubilized preparation of mouse microsomal membranes by affinity chromatography. The receptors were solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)(Chaps) a zwitterionic derivative of cholic acid. The affinity gel was prepared by coupling ovine prolactin to CH-Sepharose 4B. After extensive elution of the nonspecifically adsorbed proteins, the receptors were eluted with 4 M urea/1 M NaCl/0.5% Chaps followed by 5 M MgCl2/0.5% Chaps. The eluted receptor appeared on NaDodSO4/polyacrylamide gels as a single major band of Mr 37,000. The purified receptor retained its specificity for lactogenic hormones and binds 125I-labeled ovine prolactin with a Ka of 2-6 X 10(9) M-1.
Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.
Authors: Stetler D , Das H , Nunberg J H , Saiki R , Sheng-Dong R , Mullis K B , Weissman S M , Erlich H A
Published: May 2006

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A)+ mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR alpha chain, were used to prime a cDNA synthesis reaction on various poly(A)+ mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha mRNA was confirmed by primer extension and blot analysis. The cDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A)+ mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of cDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.
Post-translational processing of cholecystokinin in pig brain and gut.
Authors: Eng J , Shiina Y , Straus E , Yalow R S
Published: May 2006

A sequential extraction method employing methanol extraction of the COOH-terminal fragments of cholecystokinin (CCK) from pig tissues followed by HCl extraction of intact CCK and its NH2-terminal fragments is described. Radioimmunoassay of extracts and their fractionation by Sephadex chromatography and HPLC demonstrate that the distributions of COOH-terminal and NH2-terminal immunoreactivities among various regions of brain are similar and independent of the concentrations in individual regions. The distribution in gut differs from that in brain. Greatest concentrations of CCK immunoreactivity are located in cortical tissue in the brain and in duodenal mucosa in gut. Both brain and gut contain CCK octapeptide (CCK8) and an NH2-terminal fragment that is likely to be desoctapeptide-CCK33. Intact CCK33 is extractable from gut but not from brain. Brain contains another NH2-terminal immunoreactive molecule lacking COOH-terminal immunoreactivity that may be a peptide with a COOH-terminal extension, as has been described for gastrin, or one that may not be derived from a CCK33-like precursor. This peptide is much less prominent in gut, or may be nonexistent there. The failure to find CCK33 in the brain and the presence in the brain of this as-yet-uncharacterized NH2-terminal peptide raises the question as to whether the differences between neuronal and mucosal tissues are a consequence of differences in post-translational processing or in the DNA templates.
Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki.
Authors: Held G A , Bulla L A , Ferrari E , Hoch J , Aronson A I , Minnich S A
Published: May 2006

Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.
Chromatin conformational changes accompany transcriptional activation of a glucose-repressed gene in Saccharomyces cerevisiae.
Authors: Sledziewski A , Young E T
Published: May 2006

In the present study we have analyzed the kinetics of DNase I digestion of the two alcohol dehydrogenase (ADH) genes within yeast nuclei. We have found that in yeast grown on glucose the constitutively transcribed ADCI gene is much more sensitive to DNase I digestion than is the repressible ADR2 gene. In yeast grown on ethanol, both genes are transcribed and both exhibit the same sensitivity to DNase I attack. We have also found and mapped DNase I hypersensitive sites near the 5' ends of constitutive and repressible ADH genes. These sites are well correlated with the position at which transcription is initiated.
Efficient expression of Escherichia coli galactokinase gene in mammalian cells.
Authors: Schümperli D , Howard B H , Rosenberg M
Published: May 2006

The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid. Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene. This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals. Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation. In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity. Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source. The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.
Partial characterization of the mRNA that codes for enkephalins in bovine adrenal medulla and human pheochromocytoma.
Authors: Comb M , Herbert E , Crea R
Published: May 2006

[Met]enkephalin and [Leu]enkephalin are derived from a protein in bovine adrenal medulla that contains multiple copies of [Met]enkephalin [Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 3265--3268.] Here we characterize pro-enkephalin mRNA from bovine and human tissue by use of an oligodeoxynucleotide pentadecamer probe complementary to codons for Tyr-Gly-Gly-Phe-Met ([Met]enkephalin). This probe hybridizes specifically to a species of poly(A)-RNA from adrenal medulla and human pheochromocytoma, (1400--1450 bases), and also to [Met]enkephalin-containing pro-opiomelanocortin mRNAs from bovine pituitary (1200 bases) and from mouse pituitary tumor cell (1100 bases). A cloned cDNA probe (144 bases) complementary to the region of pro-opiomelanocortin mRNA that codes for lipotropin does not hybridize to the RNA from bovine adrenal medulla, demonstrating that the latter RNA is not pro-opiomelanocortin mRNA. The pentadecamer probe was extended to make cDNA with reverse transcriptase after hybridizing it to adrenal poly(A)-RNA. The sequence of an extended cDNA, 62 bases in length, was found to correspond exactly to that expected from the amino acid sequence of peptide E (a bovine adrenal peptide containing [Met]- and [Leu]enkephalin sequences). This cDNA also forms a specific hybrid with the RNA from bovine adrenal and human pheochromocytoma, confirming that these species of RNA are pro-enkephalin mRNA.
Thermosensitive development and tip regulation in a mutant of Dictyostelium discoideum.
Authors: Barclay S L , Henderson E J
Published: May 2006

A thermosensitive developmental mutant of Dictyostelium discoideum identifies a gene product that is nonessential for cell multiplication but is continuously required during aggregation and the period when multicellular mounds are formed. After mounds form a tip, which has the properties of an embryonic organizer, this gene product may be nonessential. Surgical removal of the tip from a polarized developing multicellular structure (the slug) leads to emergence of a new tip at the permissive temperature but not at the restrictive temperature. The mutant continues to develop abnormally when mixed with wild-type cells; therefore, a cell-limited rather than an exchangeable factor is altered. Assays show that the mutant has a thermosensitive defect in chemotaxis toward cAMP. The mutation reduces the number of cell surface cAMP receptors expressed at the restrictive temperature without affecting their dissociation constants or their apparent thermostability. The expression of two developmentally regulated enzymes, N-acetylglucosaminidase and cAMP phosphodiesterase, is unaffected by the mutation.
Selecting binding and complement-mediated lysis of human hepatoma cells (PLC/PRF/5) in culture by monoclonal antibodies to hepatitis B surface antigen.
Authors: Shouval D , Wands J R , Zurawski V R , Isselbacher K J , Shafritz D A
Published: May 2006

High-affinity 125I-labelled monoclonal antibodies to hepatitis B virus surface antigen (HBsAg) bind to human hepatocellular carcinoma cell line, PLC/PRF/5, which synthesizes and secretes HBsAg. These monoclonal antibodies of the IgG1, IgG2a, and IgM isotypes are directed against different antigenic determinants on HBsAg and, in the presence of complement, both anti-HBs IgG2a and IgM, but not anti-HBs IgG1, lyse PLC/PRF/5 cells in culture. Although there is a low level of anti-HBs binding (especially with anti-HBs IgM) to human hepatoma cell lines which do not synthesize HBsAg (SK-Hep 1 and Mahlavu cells), this interaction does not lead to complement-mediated cell lysis and is thought to be nonspecific. Minimal binding of 125I-labeled anti-influenza HA antigen IgM binding to PLC/PRF/5 cells was also detected, but this likewise did not lead to complement-mediated cell lysis. These results indicate that a human hepatocellular carcinoma cell line, persistently infected with hepatitis B virus, can be recognized and lysed by monoclonal antibodies directed against specific determinants of HBsAg. Monoclonal antibodies to this viral envelope protein may prove to be useful immunodiagnostic and immunotherapeutic agents when such viral epitopes are expressed on the surface of infected cells.
Opioids regulate cGMP formation in cloned neuroblastoma cells
Authors: Gwynn Germaine J. , Costa Erminio
Published: May 2006

Opioid agonists caused a rapid dose-related elevation of the cGMP content of N4TG1 murine neuroblastoma cells. An excellent correlation was found between the rank order of potency of agonists in stimulating cGMP accumulation and in displacing [
3
H]etorphine ([
3
H]ETP) bound to intact cells. The narcotic antagonists naloxone and diprenorphine failed to increase cGMP content; moreover, in the presence of 5 μM naloxone, the EC
50
of ETP increased from ≈9 nM to > 1 μM. N4TG1 cells that had been incubated for 20 min with 0.32 μM ETP and thoroughly washed displayed a marked loss in sensitivity to subsequent ETP challenge. This desensitization was characterized by a 40-50% decrease in maximal response and an increase in the apparent
K
a
of ETP from 4 to 50 nM. Desensitization was complete after a 7-min incubation with 0.32 μM ETP (
t
½
≈ 1 min) and was only slowly reversible (
t
½
> 60 min). Naloxone (5 μM) and diprenorphine (0.1 μM) failed to elicit desensitization, but they blocked ETP-induced desensitization. Dextrophan and (+)-ethylketazocine were <1% as effective as levorphanol and (-)-ethylketazocine, respectively, in both stimulating cGMP accumulation and inducing desensitization. When the binding of [
3
H]ETP (0.2-20 nM) was examined under identical experimental conditions, cells that were completely desensitized by incubation with ETP (7 min with 0.32 μM or 20 min with 15 nM) showed no loss of high-affinity recognition sites. After longer incubation with ETP (0.32 μM for 20-60 min), the maximal binding of [
3
H]ETP was reduced 17-41%. The specific short-term desensitization of cGMP accumulation is not mediated or accompanied by a decrement in the number of agonist binding sites.
On a new gap phenomenon in riemannian geometry
Authors: Greene R. E. , Wu H.
Published: May 2006

This note announces some theorems that show that the flat metric of euclidean space has no nearby nonflat complete metric with the property that its curvature keeps a sign and is “uniformly small” in a sense made precise here.
Plasminogen Tochigi: inactive plasmin resulting from replacement of alanine-600 by threonine in the active site.
Authors: Miyata T , Iwanaga S , Sakata Y , Aoki N
Published: May 2006

Structural studies on a hereditarily abnormal plasminogen, plasminogen Tochigi, have been performed to identify the difference responsible for its lack of proteolytic activity. The plasminogen sample used was from a heterozygote and thus consisted of apparently equal amounts of normal and defective plasminogen molecules. Amino acid sequence analysis of a tryptic peptide isolated from the abnormal plasminogen indicated that Ala-600 (equivalent to Ala-55 in the chymotrypsin numbering system) had been replaced by Thr. No other substitutions in the active-site residues--namely, His-57, Asp-102, and Ser-195--were found. Molecular models for chymotrypsin and the bovine trypsin-pancreatic trypsin inhibitor complex indicate that Ala-55 is very near the active-site His. The Thr at position 55 in plasminogen (plasmin) Tochigi may perturb His-57 such that the proton transfers associated with the normal catalytic process cannot occur in the abnormal plasmin.
Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.
Authors: Birktoft J J , Fernley R T , Bradshaw R A , Banaszak L J
Published: May 2006

The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms.
Evidence that a reducible xylosyl-lysine is the protein linkage of dermatan sulfate.
Authors: Longas M O , Meyer K
Published: May 2006

Dermatan sulfate, purified by standard methods, displayed one spot at the position of the standard after two-dimensional cellulose acetate electrophoresis and was 99% in GalN, 21.5% in sulfate, and 0.6% in protein; Gal and Xyl (2:1) were the only neutral sugars detected. Its glucuronic acid/iduronic acid ratio was 0.15 and its Mr was approximately equal to 16,000. On reaction with 0.4 M NaOH, its reducing group(s) determined as Glc increased by 71% with concomitant separation of protein and polysaccharide and no alteration of the amino acids; when this reaction was repeated in the presence of 0.3 M NaBH4, only 31% of Lys was detected by standard amino acid analysis and none by TLC of the dansylated amino acids; alkaline cleavage in these conditions yielded only 30% of the original Xyl, xylitol, and a ninhydrin-positive substance different from GalN, which had the retention time of xylitol on Affi-Gel 601 and was also obtained from reduction of dermatan sulfate with a 400-eq excess of Na3BH4 in water under conditions that did not cleave the dermatan sulfate-protein bond. The data indicate that a reducible xylosyl-lysine is the protein linkage of dermatan sulfate from calf ligamentum nuchae.
Tissue-specific and species-specific monoclonal antibodies to avian red cell nuclear proteins.
Authors: Kane C M , Cheng P F , Burch J B , Weintraub H
Published: May 2006

In order to identify potential red cell-specific regulatory proteins and to define additional red cell-specific markers, we have isolated a series of hybridomas that produce monoclonal antibodies that react with nuclear preparations from avian red blood cells. Several antibodies have been well characterized for their tissue- and species-specific reactions by using solid-phase and protein-transfer radioimmunoassays as well as immunofluorescence. These antibodies should allow isolation and characterization of individual nuclear proteins that are tissue and species specific and may prove useful for the study of gene expression in the erythropoietic system. The majority of the well-characterized antibodies appear to have tissue-specific properties. However, three antibodies react with all tested chicken tissues; one of these reacts with multiple peptides in a pattern that varies qualitatively and quantitatively between the tissues. This may reflect a common protein domain or modification that is used in several different tissues for similar functions but is nevertheless present in an overall protein framework that is tissue specific. Because the major fraction of the hybridomas initially produced is tissue specific, we presume that the immune system selects for tissue-specific determinants. This property of the immune system may prove to be a useful general feature of this type of analysis.
Expression of transferred thymidine kinase genes is controlled by methylation.
Authors: Christy B , Scangos G
Published: May 2006

Plasmid pTKx-1, containing the herpes simplex virus gene for thymidine kinase (TK) inserted into the BamHI site of plasmid pBR322, was introduced into Ltk- cells by calcium phosphate precipitation in the absence of carrier DNA. Line 101 is a TK+ derivative of Ltk- that contains multiple copies of pTKx-1 in a multimeric structure. A derivative of 101 that retained but no longer expressed the herpes simplex TK genes (termed 101BU1) and derivatives of line 101BU1 that reexpressed the genes (termed 101H1, 101HC, and 101HG) were selected. The TK genes in 101BU1 were hypermethylated relative to those in the TK+ parent and derivatives. Growth of 101BU1 in the presence of the methylation inhibitor 5-azacytidine resulted in an average 13-fold increase in the number of TK+ reexpressors, DNA from 101BU1 was inactive in secondary gene transfer, whereas DNA from 101 and from TK+ reexpressors was active. These data support a causative relationship between DNA methylation and decreased gene expression. All TK+ reexpressors examined had DNA rearrangements involving TK DNA.
Identification of the 45-base-long primordial building block of the entire class I major histocompatibility complex antigen gene.
Authors: Ohno S , Matsunaga T , Epplen J T , Itakura K , Wallace R B
Published: May 2006

Within the 885-base-long incomplete coding sequence for the mouse class I major histocompatibility complex (MHC) antigen H-2Kb heavy chain, we have identified 32 recurring base oligomers (1 decamer, 4 octamers, 9 heptamers, and 18 hexamers), the decamer being T-C-A-C-C-C-T-G-A-G. The compilation of these recurring base oligomers led to the identification of the 45-base-long primordial building block for this class of genes which is: C-C-T-G-C-G-G-A-C-A-A-T-G-T-C-A-C-C-C-T-G-A-G-C-T-G-C-T-G-G-G-C-T-C-T-G-G-G-C-G - T-T-G-A-C. Inasmuch as these base oligomers that recurred within the mouse H-2Kb coding sequence proper also recurred within the adjacent, transcribed noncoding sequence, we conclude that the entire approximately equal to 4,000-base-long germ-line DNA segment containing eight or nine separate exons of the coding sequence for class I MHC antigen heavy chains can be construed as having evolved from tandem repeats of this one 45-base-long primordial building block.
Gravitational field of a charged mass point
Authors: Pekeris C. L.
Published: May 2006

Adopting, with Schwarzschild, the Einstein gauge (ǀ
μν
ǀ = -1), a solution of Einstein's field equations for a charged mass point of mass
M
and charge
Q
is derived, which differs from the Reissner-Nordstrøm solution only in that the variable
r
is replaced by
R
= (
r
3
+
a
3
)
⅓
, where
a
is a constant. The Newtonian gravitational potential ψ ≡ (2/
c
2
)(1 - g
00
) obeys exactly the Poisson equation (in the
R
variable), with the mass density equal to (
E
2
/4π
c
2
),
E
denoting the electric field. ψ also obeys a second linear equation in which the operator on ψ is the square root of the Laplacian operator. The electrostatic potential Φ (=
Q/R
), ψ, and all the components of the curvature tensor remain
finite
at the origin of coordinates. The electromagnetic energy of the point charge is finite and equal to (
Q
2
/
a
). The charge
Q
defines a pivotal mass
M
*
= (
Q
/G
½
). If
M
<
M
*
, then the whole mass is electromagnetic. If
M
>
M
*
, the electromagnetic part of the mass

M
em

equals [
M
- (
M
2
-
M
*2
)
½
], whereas the material part of the mass

M
mat

equals (
M
2
-
M
*2
)
½
. When
M
>
M
*
, the constant
a
is determined, following Schwarzschild, by shrinking the “Schwarzschild radius” to zero. When
M
<
M
*
,
a
is determined so as to make the gravitational acceleration vanish at the origin.
Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex.
Authors: Nagata K , Guggenheimer R A , Enomoto T , Lichy J H , Hurwitz J
Published: May 2006

A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.
Rimorphin, a unique, naturally occurring [Leu]enkephalin-containing peptide found in association with dynorphin and alpha-neo-endorphin.
Authors: Kilpatrick D L , Wahlstrom A , Lahm H W , Blacher R , Udenfriend S
Published: May 2006

The tridecapeptide NH2-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-COOH has been purified from extracts of bovine posterior pituitary glands. This unique peptide, which has been given the name "rimorphin," is a major [Leu]enkephalin-containing peptide in all tissues examined that contain dynorphin and alpha-neo-endorphin. However, except for the initial hexapeptide sequence, it is structurally unrelated to the other two peptides.
Regulation of double-stranded RNA-activated eukaryotic initiation factor 2α kinase by type 2 protein phosphatase in reticulocyte lysates
Authors: Petryshyn Ray , Levin Daniel H. , London Irving M.
Published: May 2006

Protein synthesis initiation in reticulocyte lysates is inhibited by low concentrations (1-20 ng/ml) of double-stranded RNA (ds RNA) due to the activation of a ds RNA-dependent cAMP-independent protein kinase (ds I) that phosphorylates the α subunit of the eukaryotic initiation factor eIF-2. In lysates, ds I is present in the latent inactive form and is associated with the ribosome complement. Latent ds I is solubilized by extraction with high-salt buffers and can be purified in its latent form. Activation of purified latent ds I requires ds RNA and ATP and is accompanied by the ds RNA-dependent autophosphorylation of a polypeptide doublet of 70,000 and 72,000 daltons (“70k/72k”), which represent different phosphorylated states of the same polypeptide. These are phosphorylated in the sequence 70k→72k; increased phosphorylation of 72k is associated with increased ds I activation. Lysates (or Sepharose 6B ribosomes) treated with ds RNA display a similar ds I phosphoprotein profile, and this is accompanied by the phosphorylation of endogenous eIF-2α (38,000 daltons). Delayed
32
P pulses in ds RNA-inhibited lysates indicate that the phosphates on ds I and eIF-2α turn over. Under defined conditions, activated ds I in lysates is selectively dephosphorylated by endogenous protein phosphatase(s), and this is accompanied by the dephosphorylation of eIF-2α. Similarly, purified activated ds I is rapidly dephosphorylated by unfractionated lysate protein phosphatase(s) and by type 2 protein phosphatase but not by type 1 protein phosphatase. The dephosphorylation of ds I occurs in the sequence 72k→70k and is correlated with ds I inactivation. The heat-stable protein phosphatase inhibitor-2, which selectively blocks type 1 protein phosphatase, does not significantly affect the dephosphorylation of ds I by type 2 protein phosphatase or by unfractionated lysate phosphatases. The data support the conclusion that a ds I phosphatase activity with type 2 characteristics is involved in the regulation of ds I activity.
Mechanism of active transport: free energy dissipation and free energy transduction.
Authors: Tanford C
Published: May 2006

The thermodynamic pathway for "chemiosmotic" free energy transduction in active transport is discussed with an ATP-driven Ca2+ pump as an illustrative example. Two innovations are made in the analysis. (i) Free energy dissipated as heat is rigorously excluded from overall free energy bookkeeping by focusing on the dynamic equilibrium state of the chemiosmotic process. (ii) Separate chemical potential terms for free energy donor and transported ions are used to keep track of the thermodynamic state of each substrate through the reaction cycle. These procedures clarify the mechanism of free energy transduction, even without step-by-step analysis. The results show that free energy exchange must occur in its entirety among protein-bound species. Imposition of conditions for an adequate rate of physiological function further indicates (i) that the standard free energy of hydrolysis of protein-bound ATP (to yield protein-bound products) needs to differ substantially from the standard free energy of hydrolysis in solution and (ii) that binding sites for the transported ions must have different affinities when facing opposite sides of the membrane. The results also demonstrate that step-by-step "basic" free energy changes (often used in the form of free energy level diagrams) are inherently unsuited for analysis of the mechanism of free energy transduction.
Platelet stimulation by antifibronectin antibodies requires the Fc region of antibody.
Authors: Holderbaum D , Culp L A , Bensusan H B , Gershman H
Published: May 2006

Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody with platelet membrane fibronectin was responsible for this stimulation, we prepared proteolytic fragments of the antifibronectin antibodies. Purified F(ab')2 fragments had a greatly diminished ability to elicit degranulation, and rabbit F(ab')2 fragments were totally ineffective in this regard. Preimmune IgG from these sources was capable of inhibiting antibody-induced serotonin release in a dose-dependent manner. Purified antibody Fc fragments also inhibited this stimulation in doses stoichiometrically equivalent to the inhibition seen with preimmune IgG. These results suggest that (i) platelet stimulation elicited by anti-human fibronectin antibody is mediated by the platelet Fc receptor and therefore is likely to be dependent on antigen-antibody complex formation and (ii) fibronectin on the platelet surface may not be the primary receptor for platelet adherence to collagen.
Genetic analysis of life-span in Caenorhabditis elegans.
Authors: Johnson T E , Wood W B
Published: May 2006

Crosses between Bristol and Bergerac strains of the self-fertilizing hermaphroditic nematode Caenorhabditis elegans do not show the heterosis effects for life-span that complicate analysis of interstrain crosses with Drosophila or mice. Instead they yield F1 progeny with life-spans similar to those of the parent strains. By analysis of life-span variation among progeny F2 populations from such crosses and by two independent analyses of life-spans among recombinant inbred lines derived from F2 individuals by 18 rounds of self-fertilization, we estimate that the heritability of life-span in C. elegans is between 20% and 50%. Recombinant inbred lines show a range in mean life-spans of 10 days to 31 days compared to life-spans of about 18 days for each of the two parental strains. We conclude that life-span variation in C. elegans has a substantial genetic component and that this organism offers promising opportunities for selective breeding of longer-lived strains and genetic analysis of senescence.