bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A.
Authors: Weddell G N , Yansura D G , Dowbenko D J , Hoatlin M E , Grubman M J , Moore D M , Kleid D G
Published: May 2006

The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.
Detection in extracts of bovine brain of lipophilic complexes of sulfate esters of cholesterol and beta-sitosterol.
Authors: Prasad V V , el-Maraghy E , Ponticorvo L , Lieberman S
Published: May 2006

Evidence indicating that there exist in bovine brains hitherto-unrecognized lipophilic conjugates of sterol sulfates is presented. These conjugates are soluble in nonpolar solvents and, when heated in methanol containing pyridine, yield polar sterol conjugates. These polar substances have the chromatographic mobility of sterol sulfates and are cleaved to free sterols when subjected to a solvolytic process known to be specific for sulfate esters. The brain sterols that have been identified in this way are cholesterol and beta-sitosterol.
Pertussis toxin inhibits fMet-Leu-Phe- but not phorbol ester-stimulated changes in rabbit neutrophils: role of G proteins in excitation response coupling.
Authors: Volpi M , Naccache P H , Molski T F , Shefcyk J , Huang C K , Marsh M L , Munoz J , Becker E L , Sha'afi R I
Published: May 2006

Addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe- induced increases in Na+ influx and in intracellular pH. In addition, pretreatment of the cells with the toxin inhibits the decrease in the levels of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and the enhanced production of phosphatidic acid produced by the chemotactic factor fMet-Leu-Phe. Furthermore, the fMet-Leu-Phe-induced changes in the phosphorylation of a 46-kDa protein and of several other proteins are also inhibited by the toxin. On the other hand, the phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation of several proteins are not inhibited by the toxin. PMA, but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate, was also found to stimulate Na+ influx and to increase the intracellular pH in rabbit neutrophils. These ionic effects, like those produced by fMet-Leu-Phe, are inhibited by amiloride. The stimulated Na+ influx and H+ efflux produced by the phorbol ester, on the other hand, are not inhibited by pertussis toxin. The results reported here suggest that the activity of the Na+/H+ antiport in neutrophils is regulated by protein kinase C; that the G-protein system, either directly or indirectly, is involved in the stimulus-response coupling sequence in these cells; and that the toxin acts at, or prior to, the steps responsible for the activation of phospholipase C, and it does not affect the sequence of reactions initiated by the activation of the protein kinase C.
Purification of a vitamin K epoxide reductase that catalyzes conversion of vitamin K 2,3-epoxide to 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone.
Authors: Mukharji I , Silverman R B
Published: May 2006

An enzyme from bovine liver microsomes that catalyzes the reduction of vitamin K 2,3-epoxide to 2- and 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone was purified 1152-fold to apparent homogeneity. Microsomes were solubilized with 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the enzyme was purified by chromatography on PBE-94 ion exchanger, hydroxylapatite, and DEAE-cellulose, and then gel filtration on Sephacryl S-200. The homogeneity of the final preparation was established by polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the native enzyme is 25,000 and that of denatured enzyme is 12,400, which suggests that the enzyme is a dimer with identical subunits. No chromophoric cofactors are associated with the enzyme. Dithiothreitol and CHAPS are essential for activity, but high concentrations of glycerol reduces the activity. The enzyme is not inhibited by warfarin, a potent inhibitor of the vitamin K epoxide reductase, which catalyzes the conversion of vitamin K 2,3-epoxide to vitamin K. Evidence is presented indicating that the purified enzyme is not simply a fragment of the warfarin-sensitive vitamin K epoxide reductase.
Nucleotide sequence and organization of the mouse adenine phosphoribosyltransferase gene: presence of a coding region common to animal and bacterial phosphoribosyltransferases that has a variable intron/exon arrangement.
Authors: Dush M K , Sikela J M , Khan S A , Tischfield J A , Stambrook P J
Published: May 2006

We have determined the nucleotide sequence of a functional mouse adenine phosphoribosyltransferase (APRT) gene and its cDNA. The amino acid sequence of the enzyme is deduced from an open reading frame in the cDNA and predicts a protein with a molecular weight of 19,560. The protein coding region of the gene is approximately 2 kilobases, and it is composed of five exons and four introns. While the body of the gene is 53% G + C, the 200 nucleotides upstream from the ATG translation start codon are 66% G + C and contain three copies of the sequence C-C-G-C-C-C. The mouse APRT enzyme shares a homologous 20-amino acid sequence with mouse, hamster, and human hypoxanthine phosphoribosyltransferases (HPRTs) and several bacterial phosphoribosyltransferases. This sequence has previously been shown to be a likely catalytic domain in human HPRT and Escherichia coli glutamine phosphoribosyltransferase. Because of the similarities in function of these proteins, both eukaryotic and prokaryotic, it is not unexpected that they should exhibit one or more regions of homology, particularly at the 5-phosphoribosyl-1-pyrophosphate and purine binding sites, especially if they are related via a common evolutionary lineage. This homologous sequence is interrupted by a single intron in the mouse APRT gene and by two introns in the mouse HPRT gene. Furthermore, the positions of both introns in the HPRT sequence are different from that of the single intron in the corresponding sequence of the APRT gene.
Modified cryptochrome
in vivo
absorption in
dim
photosporulation mutants of
Trichoderma
Authors: Horwitz B. A. , Gressel J. , Malkin S. , Epel B. L.
Published: May 2006

Mutants of
Trichoderma harzianum
that are defective in blue-light-induced sporulation were induced by nutritional stresses as an alternative to light. These mutants may be modified in the putative photoreceptor pigment “cryptochrome” or in an early transduction component.
dim
(dimsighted) mutants induced by a short transient stress were mapped into four complementation groups and were chosen for study of pigment deficiencies by
in vivo
absorption spectroscopy. Mutants rib
-
10 and lys
-
44 in the
dimY
complementation group had altered
in vivo
absorption spectra in the blue region. Difference spectra obtained by subtracting
dimY
spectra from that of the wild type had difference bands with peaks at 455 and 480 nm. The similarity between the
in vivo
difference spectra and the action spectrum for sporulation in wild-type
Trichoderma
suggests that the mutants lack cryptochrome or have a defective cryptochrome. The decrease in photoresponse as well as the modification of the action spectrum near 480 nm in a
dimY
mutant support these suggestions. Both
dimY
mutants pleiotropically accumulate a yellow water-soluble pigment absorbing at wavelengths lower than the blue maxima of cryptochrome; this yellow pigment may be related to cryptochrome.
Nucleotide sequence preservation of human mitochondrial DNA.
Authors: Monnat R J , Loeb L A
Published: May 2006

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms.
Predicted protein sequence of the murine I-E-beta S-polypeptide chain from cDNA and genomic clones.
Authors: Mengle-Gaw L , McDevitt H O
Published: May 2006

Ia antigens are polymorphic cell-surface glycoprotein complexes, encoded within the I region of the mouse major histocompatibility complex, that control the ability of the organism to mount effective antigen-specific immune responses. We have isolated and determined the nucleotide sequences of cDNA and genomic clones for the I-E beta s gene and we present the predicted protein sequence for most of the E beta s polypeptide chain. The E beta s polypeptide shows 95% protein homology to the other cloned E beta alleles. Comparison of the protein sequences of five E beta alleles from haplotypes that differ in responder phenotype to pigeon cytochrome c suggests that the structure of one of the E beta hypervariable regions may determine responsiveness to this antigen.
"Exon-shuffling" maps control of antibody- and T-cell-recognition sites to the NH2-terminal domain of the class II major histocompatibility polypeptide A beta.
Authors: Germain R N , Ashwell J D , Lechler R I , Margulies D H , Nickerson K M , Suzuki G , Tou J Y
Published: May 2006

To investigate the role of the highly polymorphic amino-terminal (beta 1) domain of the class II major histocompatibility polypeptide A beta during recognition by T cells and antibodies, "exon-shuffling" was carried out between genomic recombinant DNA clones of Ak beta and Ad beta to generate a hybrid gene containing Ak beta exons for the amino-terminal domain followed by the Ad beta exons for the remainder of the molecule. L-cell gene transfectants expressing this hybrid A beta gene in combination with Ak alpha were compared to L cells expressing wild-type Ak beta Ak alpha dimers in tests of antigen-presentation to T-cell clones and hybridomas and for staining by a panel of anti-I-Ak-specific monoclonal antibodies. These antibodies were also tested for their reactivity with a B-lymphoma transfectant expressing Ak beta in the absence of Ak alpha. The results showed no qualitative differences in either T-cell or antibody-mediated recognition of I-Ak molecules containing either the exon-shuffled or wildtype Ak beta. Together with the data involving the B cell transfectant expressing only Ak beta, these results map control of the A beta contribution to the immunologically relevant determinants of I-Ak to the highly polymorphic amino-terminal domain and indicate little, if any, contribution to allele-specific recognition by amino acid sequence variations in the remaining portions of the A beta polypeptide.
Antigen-binding specificities of antibodies are primarily determined by seven residues of VH.
Authors: Ohno S , Mori N , Matsunaga T
Published: May 2006

Although the antigen-binding pocket of all antibodies consists of VL + VH dimers (where VL and VH represent immunoglobulin light and heavy chain regions, respectively), subgroups of their VH largely determine their antigen-binding specificities. This VH subgroup dependence automatically relegates subsidiary roles to VL as a whole and to the complementarity-determining region 3 (CDR-3) of VH encoded by independent diversity (D) and joining (J) coding segments in determining antigen-binding specificities of individual antibodies. As a sequel to our previous paper, which emphasized the role conserved residues in CDR-1 and CDR-2 of VH play in general shaping of the primordial antigen-binding cavity, here we propose that the three short clusters of amino acid sequences in CDR-1 and CDR-2 that are placed in the immediate vicinity of the tryptophan loop primarily determine subgroup-dependent antigen preference of individual VH, therefore, antibodies. The three clusters are the 31st to 35th positions of CDR-1 and the 50th to 52nd and 58th to 60th positions of CDR-2. Of those, the 32nd, 34th, 51st, and 59th positions tend to be occupied by tyrosine, methionine, isoleucine, and tyrosine, respectively. Nevertheless, free amino acid substitutions at the remaining seven sites can generate 20(7) or 1.28 X 10(9) varieties of amino acid sequence combinations. Some of these astronomically numerous sequence combinations no doubt contribute to the maintenance of the vast repertoire of antigen-combining diversity, which might be as large as 10(7), whereas others serve to vary binding affinities toward the same antigen. Ironically, but not surprisingly, a single nonconservative amino acid substitution at one of these sites often suffices to change the antigen preference of VH from one to another, whereas more substitutions affecting two or more clusters are apparently required to change the binding affinity toward the same antigen. In the case of mouse anti-p-azophenylarsonate antibodies, the principle of VH subgroup dependence is violated, their VH belonging to either subgroup 1 or 3. It appears that the mouse genome lacks anti-p-azophenylarsonate germ line VH, residues of CDR-3 derived from one particular JH coding segment coming to rescue to cope with this unnatural man-made antigen.
O6-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.
Authors: Brent T P , Houghton P J , Houghton J A
Published: May 2006

Immune-deprived female CBA/CaJ mice bearing xenografts of six different human rhabdomyosarcoma lines were treated with 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU). Tumor responses were compared with levels of O6-methylguanine-DNA methyltransferase activity because of evidence indicating that repair of DNA interstrand cross-link precursors, mediated by the transferase, may be an important determinant of MeCCNU cytotoxicity. Levels of methyltransferase in tumor extracts were measured by determining the loss of O6-methylguanine from 3H-labeled methylated DNA. Five of the six tumor lines examined showed either no response to MeCCNU or regrowth after an incomplete response. In each instance, the extent of tumor regression correlated with the level of O6-methylguanine-DNA methyltransferase activity in tumor extracts. The single highly drug sensitive line was totally devoid of the activity. These results suggest that O6-methylguanine-DNA methyltransferase levels in human tumor cells may be a clinically useful predictor of sensitivity to the chloroethylnitrosoureas.
Natural history of restricted synthesis and expression of measles virus genes in subacute sclerosing panencephalitis.
Authors: Haase A T , Gantz D , Eble B , Walker D , Stowring L , Ventura P , Blum H , Wietgrefe S , Zupancic M , Tourtellotte W
Published: May 2006

Subacute sclerosing panencephalitis (SSPE) is a slow infection caused by measles virus in which several years separate recovery from typical acute measles and the development of a slowly progressive neurological disease. We have investigated replication of measles virus in brain tissue obtained after the onset of neurological disease and in the terminal phase. With a hybridization tomographic technique that combines in situ hybridization with macroradioautographic screening of large areas of tissue, we analyzed the spatial and temporal distribution of virus genes in vivo, using region- and strand-specific probes for the nucleocapsid and matrix genes. We show that early in the course of SSPE there is a global repression in the synthesis and expression of the genome. In the final stage of SSPE most infected cells still have depressed levels of plus- and minus-strand viral RNA and contain nucleocapsid protein but lack matrix protein. These findings provide further evidence for a unified view of slow infections of the nervous system, where the general constraints on virus gene expression provide an explanation for persistence of virus in the face of the host's immune response, and the slow evolution of pathological change. In the final phases of SSPE the more specific block in virus replication accounts for the cell-associated state of the virus and the difficulty in virus isolation.
Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis.
Authors: Perkins J B , Youngman P J
Published: May 2006

A derivative of Tn917 was constructed, referred to as Tn917-lac, which is capable of generating fusions that connect the transcripts of Bacillus subtilis chromosomal genes to the coding sequence of the lacZ gene of Escherichia coli. Two independent insertions of Tn917-lac into the gltA gene and one insertion into the trpE gene (in the trpEDCFBA operon) of B. subtilis were studied in detail, and the results confirmed that Tn917-lac-mediated transcriptional fusions produce levels of beta-galactosidase that reflect accurately the regulated expression of interrupted genes. To facilitate these studies, a procedure was developed that permits the analysis of Tn917-lac-mediated fusions in partial diploids where insertional mutations are complemented by an intact copy of the interrupted genes. Tn917 is known to function efficiently in bacteria representing three quite different Gram-positive genera (Streptococcus, Bacillus, and Staphylococcus) and is known to display a relatively high degree of randomness in its insertions into bacterial genomes, making it likely that Tn917-lac will be useful for the identification and study of many kinds of regulated genes in a wide range of Gram-positive species.
Viroid processing: a model involving the central conserved region and hairpin I.
Authors: Diener T O
Published: May 2006

A model is proposed for the processing of oligomeric viroid replication intermediates into monomeric, circular progeny viroids. The model identifies a thermodynamically extremely stable base-paired configuration that partially or completely dimeric, as well as higher, viroid oligomers can assume and postulates that this structure, which involves structural features common to all viroids (the central conserved region and secondary hairpin I), is essential for precise cleavage and ligation. The model explains why recombinant plasmids containing tandem repeats of two or more viroid sequence equivalents are highly infectious when inoculated into viroid-susceptible plants, why certain plasmids containing partially duplicated viroid-specific inserts are less infectious, and why plasmids containing monomeric inserts are noninfectious or at best marginally infectious. The model also accounts for the fact that vector-derived sequences on either or both sides of the viroid sequence(s) of a restriction fragment are precisely excised and are lacking in progeny viroids.
The galaxy luminosity function and the redshift-distance controversy (A Review)
Authors: Salpeter E. E. , Hoffman G. L.
Published: May 2006

The mean relation between distance and redshift for galaxies is reviewed as an observational question. The luminosity function for galaxies is an important ingredient and is given explicitly. We discuss various observational selection effects that are important for comparison of the linear and quadratic distance-redshift laws. Several lines of evidence are reviewed, including the distribution of galaxy luminosities in various redshift ranges, the luminosities of brightest galaxies in groups and clusters at various redshifts, and the Tully-Fisher correlation between neutral hydrogen velocity widths and luminosity. All of these strongly favor the linear law over the quadratic.
Human inhibitor of the first component of complement, C1: characterization of cDNA clones and localization of the gene to chromosome 11.
Authors: Davis A E , Whitehead A S , Harrison R A , Dauphinais A , Bruns G A , Cicardi M , Rosen F S
Published: May 2006

C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (C1) by inactivation of the serine protease subcomponents, C1r and C1s. C1 inhibitor cDNA clones have been isolated, and one of these (pC1INH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with alpha 1-antitrypsin and alpha 1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of BamHI-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11.
Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells.
Authors: Nir U , Walker M D , Rutter W J
Published: May 2006

Two cis-acting elements, the enhancer and the promoter, independently contribute to the cell-specific expression of the rat insulin 1 gene. The activities of these elements are presumably mediated by trans-acting factors. We have performed intracellular competition experiments that suggest the presence of a negative factor(s) that represses the enhancer activity in cells that do not express the insulin gene. In these experiments fibroblast cells (COS-7) were transfected with two plasmids: a test plasmid containing the gene for chloramphenicol acetyltransferase under the control of the thymidine kinase promoter and the insulin enhancer; and a competitor plasmid containing insulin enhancer sequences and the simian virus 40 origin of replication to permit its replication in the recipient cells. The presence of the competitor plasmid led to a 5- to 6-fold increase in chloramphenicol acetyltransferase activity as compared with the activity detected when insulin enhancer was absent from either the competitor or the test plasmid. A 5-fold increase in chloramphenicol acetyltransferase activity was also seen when the rat amylase enhancer was present on the competitor plasmid; in contrast the simian virus 40 enhancer exerted no effect. Efficient derepression required additional sequences downstream from those essential for enhancer activity. We propose that the activity of the rat insulin 1 enhancer is modulated by a negative trans-acting factor(s) that is active in cells not expressing insulin but is overridden by the dominant positive trans-acting factor(s) present in insulin-producing cells.
Tumor cell autocrine motility factor.
Authors: Liotta L A , Mandler R , Murano G , Katz D A , Gordon R K , Chiang P K , Schiffmann E
Published: May 2006

A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells. We term this activity "autocrine motility factor" (AMF). AMF has the properties of a protein with an estimated size of 55 kDa. At concentrations of 10 nM or less, AMF stimulated the random or directed motility of the producer cells. However, AMF is not an attractant for neutrophils. Amino acid analysis of the purified AMF protein revealed a high content of serine, glycine, glutamic acid, and aspartic acid residues. The activity of AMF was not replaced or blocked by known growth factors such as epidermal growth factor or type beta transforming growth factor. Mechanistic studies showed that AMF stimulated the incorporation of [3H]methyl into cell membrane phospholipids after incubation with [methyl-3H]methionine with a sustained increase in the methylation of phosphatidyldimethylethanolamine to phosphatidylcholine. In contrast, AMF did not affect the incorporation of [1,2-14C]choline into phosphatidylcholine. AMF was produced in large amounts by three different clones of ras oncogene-transfected metastatic NIH 3T3 cells but not by the nontransformed parental cells. AMF may play a major role in the local invasive behavior of tumor cells and may also facilitate the concerted invasion by groups of tumor cells.
Membrane phospholipid asymmetry as a determinant of erythrocyte recognition by macrophages.
Authors: McEvoy L , Williamson P , Schlegel R A
Published: May 2006

To assess the role of transbilayer phospholipid distribution in the recognition and phagocytosis of erythrocytes by macrophages, human erythrocytes with either a symmetric or asymmetric distribution of membrane phospholipids were prepared by hypotonic hemolysis and then incubated with cultures of human monocyte-derived macrophages. Erythrocytes with an abnormal, symmetric distribution were phagocytosed 4 times more readily than their counterparts with an asymmetric distribution or than normal, asymmetric intact erythrocytes. This enhanced phagocytosis correlated with two biophysical properties of the membrane: the spacing of phospholipids, as assessed by binding of the dye merocyanine 540, and the relative hydrophobicity, as measured by aqueous two-phase polymer partitioning. These results suggest a mechanism by which loss of membrane asymmetry is translated into recognition by macrophages and provide guidelines in loading erythrocytes that may be useful in manipulating the mode of delivery when erythrocytes are used as drug carriers in vivo.
Protease and cyclooxygenase inhibitors synergistically prevent activation of human platelets.
Authors: Ruggiero M , Lapetina E G
Published: May 2006

Thrombin induces platelet aggregation and formation of a fibrin clot in platelet-rich plasma; leupeptin, a protease inhibitor, partially inhibits platelet aggregation, but it does not inhibit fibrin clot formation. Indomethacin does not inhibit either thrombin-induced platelet aggregation or fibrin clot formation. However, when the two drugs are given together, a synergistic inhibition of thrombin-induced platelet aggregation occurs, while fibrin clot formation remains unaffected. Thrombin-induced stimulation of the release of serotonin in washed human platelets is also synergistically inhibited by the combined actions of leupeptin and indomethacin. Thrombin and collagen, added simultaneously, induce full platelet aggregation and release of serotonin. Neither leupeptin nor indomethacin inhibits platelet responses elicited by both agonists; however, when leupeptin and indomethacin are given together, a synergistic inhibition of thrombin- and collagen-induced response is observed. These findings might be relevant in prophylaxis and treatment of thromboembolic disease.
Class I histocompatibility antigens and insulin receptors: evidence for interactions.
Authors: Phillips M L , Moule M L , Delovitch T L , Yip C C
Published: May 2006

We provide evidence for an interaction between mouse class I major histocompatibility complex antigens and insulin receptors. Antibodies against class I but not class II major histocompatibility complex antigens immunoprecipitate photoaffinity-labeled hepatic insulin receptors. Haplotype specificity is demonstrated by reciprocal precipitation using anti-class I antibodies and three strains of mice. Antibodies against the 45-kDa products of either the H-2K or H-2D locus and rabbit anti-mouse beta 2-microglobulin antibodies were shown to precipitate insulin receptors. We also demonstrate the specific binding of 125I-labeled insulin and 125I-labeled epidermal growth factor, but not 125I-labeled glucagon or 125I-labeled atrial natriuretic factor, to solubilized plasma membranes immunoprecipitated with anti-H-2K antibody. These observations suggest a specific interaction between class I major histocompatibility complex antigens and certain hormone receptors.
Protease-induced immunoregulatory activity of platelet factor 4.
Authors: Katz I R , Thorbecke G J , Bell M K , Yin J Z , Clarke D , Zucker M B
Published: May 2006

Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity.
Fusion of renal epithelial cells: a model for studying cellular mechanisms of ion transport.
Authors: Oberleithner H , Schmidt B , Dietl P
Published: May 2006

The investigation of epithelial ion transport at the cellular level by means of electrophysiological techniques is hampered by the small size of epithelial cells. Moreover, interpretation of experiments is complex due to poorly defined and highly variable paracellular leaks (shunt pathways). In search of a new experimental approach we developed a technique to isolate renal epithelial cells (diameter approximately equal to 10 micron) from diluting segments of the frog kidney and to fuse them to "giant" cells (diameter approximately equal to 100 micron). These cells generate membrane potentials of -54.1 +/- 1.6 mV (mean +/- SEM; n = 40). They are sensitive to the diuretic drugs furosemide and amiloride and to the K+- and Cl- -permeability blockers Ba2+ and anthracene-9-carboxylic acid. The experiments demonstrate membrane potential measurements in cells isolated from renal epithelium and fused to giant cells. The cells retain their specific membrane properties and could serve as a valuable experimental model in epithelial research.
Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription.
Authors: Ikeda R A , Richardson C C
Published: May 2006

Promoters for T7 RNA polymerase have a highly conserved sequence of 23 continuous base pairs located at position -17 to +6 relative to the initiation site for the RNA. The complex of T7 RNA polymerase with the phage phi 10 promoter has been visualized indirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA X Fe(II). The DNA contacts made by T7 RNA polymerase have been mapped during binding and during the subsequent initiation of transcription. The RNA polymerase alone protects 19 bases in a region from -21 to -3. Synthesis of the trinucleotide r(GGG) expands the length of the sequence protected by the RNA polymerase and stabilizes the complex; 29 bases (-21 to +8) are protected, and the observed equilibrium association constant of the r(GGG) complex is 5 X 10(5) M-1. The formation of a hexanucleotide mRNA, r(GGGAGA), further extends the protected region; 32 bases (-21 to +11) are protected. Finally, the synthesis of a pentadecanucleotide mRNA leads to a translocation of the region protected by the protein; the sequence now protected is reduced to 24 bases (-4 to +20).
Increased expression of p53 protein in human leukemia cells.
Authors: Koeffler H P , Miller C , Nicolson M A , Ranyard J , Bosselman R A
Published: May 2006

We examined synthesis of the cellular phosphoprotein p53 in fresh bone marrow or peripheral blood cells from normal donors and from patients with leukemia, preleukemia, or other hematopoietic disorders. Lysates of cells labeled with [35S]methionine were immunoprecipitated with monoclonal antibodies to p53, and the immunoprecipitates were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Bone marrow or peripheral blood cells from 8 of 33 patients with hematopoietic disorders showed increased p53, seven of the eight occurring in cells of patients with preleukemia or acute myelogenous leukemia. Increased p53 synthesis was not associated with p53 gene amplification, as shown by Southern blot analysis. Synthesis of p53 was not increased in any of nine normal human bone marrow samples or eight normal human peripheral blood granulocyte, macrophage, and lymphocyte samples. The hematopoietic cells of patients in remission or with chronic forms of leukemia did not generally synthesize elevated levels of p53. In addition, we found negligible p53 mRNA and protein expression in a variety of human myeloid leukemia lines blocked at different stages of differentiation. Southern blot analysis showed that, except for the HL-60 cells, the p53 gene of the myeloid cell lines was intact. In view of recent evidence implicating p53 in transformation of cultured cells, our results using fresh leukemia cells suggest that p53 may contribute to the phenotype of certain leukemias in vivo.
Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA.
Authors: Zamecnik P C , Goodchild J , Taguchi Y , Sarin P S
Published: May 2006

The possibility of using oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-III) [the etiological agent of acquired immunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P-labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynucleotides at 20 microM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 microM. Oligodeoxynucleotides complementary to a region close to the tRNALys primer binding site on HTLV-III RNA and others complementary to HTLV-III mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynucleotides suggests that the antiviral effects were specific. Although these results pertain to HTLV-III-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynucleotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex.
Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease.
Authors: Shii K , Roth R A
Published: May 2006

Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.
Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells.
Authors: Fukami Y , Nakamura T , Nakayama A , Kanehisa T
Published: May 2006

Calmodulin, a wide-spread eukaryotic Ca2+-binding protein, was phosphorylated at its tyrosine residues in Rous sarcoma virus (RSV)-transformed chicken and rat cells but not in normal chicken embryo fibroblasts. In contrast, serine and threonine phosphorylation of calmodulin was found to occur in both normal and virus-transformed cells. In an in vitro system containing purified src kinase from RSV-transformed cells, tyrosine phosphorylation of calmodulin by the src kinase was inhibited by Ca2+. Furthermore, the tyrosine-phosphorylated calmodulin showed slower mobility than that of nonphosphorylated calmodulin in NaDodSO4/polyacrylamide gel electrophoresis when Ca2+ was present. These results suggest that the structure of calmodulin Ca2+ complex may be altered by tyrosine phosphorylation. It is thus inferred that Ca2+ may regulate the level of tyrosine phosphorylation of calmodulin in RSV-transformed cells, and phosphorylation in turn may attenuate the function of this protein in vivo.
Structure of component B (7-mercaptoheptanoylthreonine phosphate) of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum.
Authors: Noll K M , Rinehart K L , Tanner R S , Wolfe R S
Published: May 2006

Component B, the heat-stable low-molecular-weight cofactor required for methane production by dialyzed cell-free extracts of Methanobacterium thermoautotrophicum, has been purified to homogeneity and its structure assigned. Results of low-resolution fast-atom-bombardment and field-desorption mass spectrometry indicated a molecular weight of 419, and high-resolution fast-atom-bombardment mass spectrometry agreed with the molecular formula C13H26NO8PS2. Evidence from fast-atom-bombardment and field-desorption mass spectrometry and 360-MHz 1H NMR in deuterium oxide argued that the compound was isolated as a mixed disulfide with 2-mercaptoethanol; so the proposed elemental formula of the free acid, free thiol would be C11H22NO7PS (molecular weight, 343). The proposed structure for an active form of the coenzyme is 7-mercaptoheptanoylthreonine phosphate.
Rapid assay of foreign gene expression in leaf discs transformed by
Agrobacterium tumefaciens
: Role of T-DNA borders in the transfer process
Authors: Horsch R. B. , Klee H. J.
Published: May 2006

We have developed a sensitive leaf disc transformation procedure for studying early and/or transient T-DNA expression during
Agrobacterium tumefaciens
-mediated transformation of plant cells. Using this system, we have examined the function of T-DNA border sequences on the early expression of T-DNA genes and on the stable integration of those genes in infected cells. Deletion of the right border from the T-DNA appears to permit transfer of T-DNA genes from the tumor-inducing (Ti) plasmid but greatly reduces the frequency of their stable integration. A binary vector has been constructed to permit examination of T-DNA border function in
trans
to the Ti plasmid. In this situation, a single T-DNA border is necessary for early expression of T-DNA genes and is sufficient for stable integration in any orientation.
Keratinocyte-derived T-cell growth factor: a T-cell growth factor functionally distinct from interleukin 2.
Authors: Kupper T S , Coleman D L , McGuire J , Goldminz D , Horowitz M C
Published: May 2006

T-cell growth factor, more recently termed interleukin 2 (IL-2), is the product of activated T lymphocytes and is considered the principal trophic factor for T lymphocytes. The activity of IL-2 preparations is assessed by the degree to which they support the growth of various IL-2-dependent cell lines. We report that murine epidermal epithelial cells (keratinocytes) produce and release a factor that supports the growth of the helper-T-cell-derived, IL-2-dependent cell line HT-2. This substance, keratinocyte-derived T-cell growth factor (KTGF), does not support the growth of an IL-2-dependent cell line derived from cytotoxic T cells (line CTLL-2). This differential effect on IL-2-dependent cell lines is unique to KTGF. KTGF has an apparent molecular weight of 25,000-35,000 and has properties similar to those of conventional IL-2 by reversed-phase and gel-filtration HPLC analysis. However, even highly purified KTGF fails to stimulate the proliferation of CTLL-2 cells. The observation that epidermal epithelium produces a trophic factor for T lymphocytes may help explain the basis for preferential proliferation of T cells in the microenvironment of skin in certain dermatologic disorders. Further, it suggests that different IL-2-dependent T-cell lines may have distinct growth requirements and that non-lymphocyte cell types may produce factors capable of maintaining the growth of T cells.
Mechanism for photosynthetic O2 evolution.
Authors: Brudvig G W , Crabtree R H
Published: May 2006

We present a mechanism for photosynthetic O2 evolution based on a structural conversion of a Mn4O6 "adamantane"-like complex to a Mn4O4 "cubane"-like complex. EPR spectral data obtained from the S2 state of the O2-evolving complex are characteristic of a Mn4O4 cubane-like structure. Based on this structure for the manganese complex in the S2 state as well as a consideration of the other evidence available on the natural system and the coordination chemistry of manganese, structures are proposed for the five intermediate oxidation states of the manganese complex. A molecular mechanism for the formation of an O--O bond and the displacement of O2 from the S4 state is easily accommodated by the proposed model. The model is discussed in terms of recent EPR, x-ray, and UV spectral data obtained from the manganese site in the photosynthetic O2-evolving complex.
Isolation of ras GTP-binding mutants using an in situ colony-binding assay.
Authors: Feig L A , Pan B T , Roberts T M , Cooper G M
Published: May 2006

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.
A repetitive DNA family (Sau3A family) in human chromosomes: extrachromosomal DNA and DNA polymorphism.
Authors: Kiyama R , Matsui H , Oishi M
Published: May 2006

In this paper, we report a tandemly repeated DNA sequence found in human chromosomes. The DNA sequence, which is present at approximately 1000 copies per haploid genome, consists of a basic unit 849 base pairs (bp) long with a single specific restriction enzyme (Sau3AI) cutting site. The unit is further composed of five subunits, each approximately 170 bp long. When DNAs from various sources were examined by Southern hybridization using the repetitive DNA as a probe, a considerable degree of restriction fragment length polymorphism was observed. Furthermore, a substantial percentage (approximately 1.0%) of the same DNA sequence was also found extrachromosomally in the cultured human (HeLa) cells as monomers and oligomers of the basic unit in the form of covalently closed circular DNA. These results suggest that the repetitive DNA is unstable and prone to be excised from the chromosomes through homologous recombination.
Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation.
Authors: Holt J T , Gopal T V , Moulton A D , Nienhuis A W
Published: May 2006

Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human beta-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.