bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels in rat liver.
Authors: Clarke C F , Edwards P A , Lan S F , Tanaka R D , Fogelman A M
Published: May 2006

Addition of cholestyramine or cholestyramine plus mevinolin to the diet has been reported to increase the activity and mass of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The present data show that these same dietary manipulations cause an induction of functional reductase mRNA. RNA was isolated from rat livers and added to an in vitro translation system, and the reductase was immunoprecipitated and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. One protein was specifically immunoprecipitated and was found to have a Mr of 90,000 on 0.5 M urea/sodium dodecyl sulfate/polyacrylamide gels and a Mr of 94,000 on 8 M urea/sodium dodecyl sulfate/polyacrylamide gels. In animals fed rat chow supplemented with 5% cholestyramine and 0.1% mevinolin, reductase mRNA levels were 5.7-fold higher than in animals fed rat chow with 5% cholestyramine and were 16-fold higher than in animals fed rat chow with 5% cholestyramine and given mevalonolactone by stomach intubation. RNA isolated from animals fed a normal diet and killed at the nadir of the diurnal cycle of enzyme activity contained no detectable amounts of reductase mRNA as determined by this assay.
Putative introns in tRNA genes of prokaryotes.
Authors: Kaine B P , Gupta R , Woese C R
Published: May 2006

Sequences of two putative tRNA genes, for serine and leucine, from the archaebacterium Sulfolobus solfataricus contain intervening sequences in the anticodon region. Furthermore, the genes lack encoded CCA 3' termini and are flanked by A + T-rich DNA segments. The introns can both form the same secondary structure, which is a double-helical extension of the anticodon stalk. The resulting structure contains two symmetrically placed 3-base bulge loops, in which are located cleavage sites for the introns. In the one case tested, the gene occurs as a single copy in the genome.
Identification of a structural gene for a RNA polymerase II polypeptide in Drosophila melanogaster and mammalian species.
Authors: Ingles C J , Biggs J , Wong J K , Weeks J R , Greenleaf A L
Published: May 2006

Using subclones representing 14 kilobase pairs (kb) of DNA from the Drosophila melanogaster RNA polymerase II (EC 2.7.7.6) X-linked genetic locus, RpII, we have identified four poly(A)+ RNA transcripts in adult flies. The DNA encoding only one of these, a 7-kb transcript, cross-hybridized to mammalian DNA. DNA from alpha-amanitin-resistant (AmaR) Chinese hamster ovary (CHO) and human cells was used to transform the temperature-sensitive (TS) RNA polymerase II Syrian hamster mutant TsAF8. The acquisition of the TS+ AmaR RNA polymerase II phenotype was accompanied by the appearance of donor-DNA-specific restriction fragments that cross-hybridize to the D. melanogaster 7-kb transcript DNA. This D. melanogaster DNA and the related DNA detected in mammalian species must therefore be the structural gene for a RNA polymerase II polypeptide.
Regulation of murine IgE production: importance of a not-yet-described T cell for IgE secretion demonstrated in SJA9 mice.
Authors: Hirano T , Kumagai Y , Okumura K , Ovary Z
Published: May 2006

To investigate why SJA9 mice do not produce IgE antibody, we took advantage of the adoptive transfer method and the carrier effect. Antibody-producing B cells (donor I) were primed with 2,4-dinitrophenylated ovalbumin (DNP-OVA) and carrier-specific T cells (donor II) were primed with keyhole limpet hemocyanin (KLH). The recipients were challenged with DNP-KLH. When 0.2 micrograms of DNP-OVA was used and the donor II cells were from SJL mice the recipient produced anti-DNP IgE. When donor I cells were from the SJL strain and donor II cells were from the SJA9 strain the recipient produced anti-DNP IgE. This pointed to a T-cell defect (absent or functionally inactive T epsilon o cell) in the SJA9 strain, which could be corrected either by injecting unprimed T cells from the SJL strain into the SJA9 donor I or by infection with Nippostrongylus brasiliensis, a hookworm, after priming. It seems that in the SJA9 strain in the absence of functioning T epsilon o cells isotype-specific (IgE) suppression, described previously in the SJL strain, is more pronounced, because when the SJA9 donor I was primed with 1 microgram of DNP-OVA only trace amounts of anti-DNP IgE were produced with SJL donor II cells.
A monoclonal antibody-enzyme immunoassay for serum carcinoembryonic antigen with increased specificity for carcinomas.
Authors: Hedin A , Carlsson L , Berglund A , Hammarström S
Published: May 2006

A two-site monoclonal antibody-enzyme immunoassay (MEIA) for carcinoembryonic antigen (CEA) was developed that uses two monoclonal anti-CEA antibodies, which recognize two different epitopes in the peptide moiety of CEA. The assay was sensitive to 0.5 micrograms/liter and had a measuring range of 0.5-200 micrograms of CEA per liter. It was highly specific inasmuch as none of three known CEA-related substances, "nonspecific crossreacting antigens 1 and 2" (NCA-1 and NCA-2) and biliary glycoprotein I (BGP I), reacted in the assay. NCA-2 (meconium antigen) is very similar to CEA. None of five commercially available CEA assays were able to differentiate between CEA and NCA-2. With one exception (colon), normal tissue extracts did not react in the MEIA even when tested at very high concentrations. Sera from a total of 180 healthy individuals and patients with malignant and nonmalignant disease were analyzed for CEA levels by using the MEIA and in parallel a conventional radioimmunoassay. A significant increase in specificity for carcinomas was obtained with the MEIA. This was essentially due to a decrease of MEIA CEA values in sera from patients with nonmalignant disease. The CEA values in the group of carcinoma patients (colon, pancreas, lung, and breast) were the same in the two assays.
Analysis of a transposable element in Caenorhabditis elegans.
Authors: Liao L W , Rosenzweig B , Hirsh D
Published: May 2006

A transposable element, designated Tc1, has been characterized in Caenorhabditis elegans. Tc1 is 1.7 kilobases long, has an inverted terminal repeat of less than 100 base pairs, and is repeated as a highly conserved element. The copy number and genomic positions of Tc1 are extremely variable among strains, implying that Tc1 is mobile. However, progeny of interstrain crosses did not show hybrid dysgenic traits that might be due to Tc1 transposition.
Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2.
Authors: Guiney D G , Yakobson E
Published: May 2006

The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the single-strand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.
Active site of RNase: neutron diffraction study of a complex with uridine vanadate, a transition-state analog.
Authors: Wlodawer A , Miller M , Sjölin L
Published: May 2006

A complex of RNase A with a transition-state analog, uridine vanadate, has been studied by a combination of neutron and x-ray diffraction. The vanadium atom occupies the center of a distorted trigonal bipyramid, with the ribose oxygen O2' at the apical position. Contrary to expectations based on the straightforward interpretation of the known in-line mechanism of action of RNase, nitrogen NE2 of histidine-12 was found to form a hydrogen bond to the equatorial oxygen O8, while nitrogen NZ of lysine-41 makes a clear hydrogen bond to the apical oxygen O2'. Nitrogen ND1 of histidine-119 appears to be within a hydrogen-bond distance of the other apical oxygen, O7. Two other hydrogen bonds between the vanadate and the protein are made by nitrogen NE2 of glutamine-11 and by the amide nitrogen of phenylalanine-120. The observed geometry of the complex may necessitate reinterpretation of the mechanism of action of RNase.
Site-specific antibodies to human erythropoietin directed toward the NH2-terminal region.
Authors: Sue J M , Sytkowski A J
Published: May 2006

Site-specific antibodies to human erythropoietin have been raised in rabbits immunized with a synthetic polypeptide composed of the putative 26 NH2-terminal amino acids of the hormone. The immunogenic peptide was coupled to bovine serum albumin. Antibodies specific for peptide were detected by enzyme-linked immunosorbent assay. They immunoprecipitated both highly purified 125I-labeled erythropoietin and biologically active erythropoietin. The immunoprecipitation of 125I-labeled erythropoietin was inhibited by unlabeled erythropoietin and by peptide, demonstrating their crossreactivity. The antibodies did not neutralize erythropoietin's biological activity. These results indicate that a portion of the NH2-terminal region of erythropoietin is exposed on the surface of the protein at some distance from the receptor-binding domain. These antibodies will be important in further studies of the hormone and its mechanism of action.
Detection of HLA-D/DR-related DNA polymorphism in HLA-D homozygous typing cells.
Authors: Owerbach D , Lernmark A , Rask L , Peterson P A , Platz P , Svejgaard A
Published: May 2006

Sequences of different sizes are generated when DNA from homozygous HLA-Dw/DR typing cells are digested with restriction endonuclease and analyzed by hybridization with a HLA-D region class II antigen beta-chain cDNA probe. The patterns of hybridization were highly polymorphic but one endonuclease, BamHI, defined sequences unique to all HLA-Dw/DR specificities 1-8 except HLA-Dw/DR 2 and 6; however, these two specificities were resolved with the enzyme EcoRI. Digestion with other endonucleases such as Pst I results in patterns of restriction fragments that differ between homozygous typing cells of the same HLA-Dw/DR specificity. HLA-D region beta-chain probes permit HLA-D region genotyping at the DNA level and may allow detection of genes controlling the association of HLA specificities with a wide variety of diseases.
Human immunoglobulin allotypes: previously unrecognized determinants and alleles defined with monoclonal antibodies.
Authors: Zelaschi D , Newby C , Parsons M , van West B , Cavalli-Sforza L L , Herzenberg L A , Herzenberg L A
Published: May 2006

The highly polymorphic system of serologically defined genetic markers on human IgG heavy chains (Gm allotypes) is second only to the HLA complex in terms of the large number of determinants, alleles, and haplotypes that can be used for analyses of disease associations and other genetic studies. However, present typing methods are based on the use of anti-Gm antisera that are derived mainly from fortuitously immunized human donors, often requiring processing before use, and must be used in a hemagglutination-inhibition assay that cannot be used in typing for isoallotypic determinants (currently termed "non-markers"). In studies presented here, we describe an allotyping system that utilizes monoclonal antibodies in a "sandwich" modification of the solid-phase radioimmunoassay, which is capable of reliable quantitative typing of allotypic, isoallotypic, and isotypic immunoglobulin determinants. We show that these highly reproducible, easily disseminated, and essentially inexhaustible reagents can be used for rapid, sensitive, and quantitative Gm typing. Using this system we define two previously unrecognized Gm determinants, one of which, found to date only in Caucasians, is different from all known Gm markers and thus defines previously unrecognized alleles and haplotypes. The other determinant co-segregates with the conventional G3m(b1) marker but is distinct from that marker on serological grounds. The successful preparation of mouse monoclonal antibodies that detect human Gm allotypic differences and the development of an assay system capable of typing isoallotypic as well as allotypic determinants opens the way to further dissection and application of this rich genetic system.
Macrophage activation: priming activity from a T-cell hybridoma is attributable to interferon-gamma.
Authors: Pace J L , Russell S W , Schreiber R D , Altman A , Katz D H
Published: May 2006

Antiviral and macrophage-priming activities in the supernatant medium of a subclone of a concanavalin A-stimulated mouse T-cell hybridoma were investigated. The two activities were associated with a molecular weight of approximately 50,000 and could not be separated by various approaches. Both activities were eliminated by a highly specific neutralizing antibody against mouse interferon-gamma, but not by antibody against interferon-alpha and -beta. The ratio of priming to antiviral activity in the hybridoma culture supernate was indistinguishable from the ratio obtained with mouse interferon-gamma prepared by recombinant DNA technology. It was concluded from these data that the priming activity in hybridoma culture supernates was attributable to interferon-gamma and that this mediator is one form of the lymphokine macrophage-activating factor. Interferon-gamma was greater than 800 times more efficient at priming mouse macrophages for tumor cell killing than was a mixture of interferon-alpha and -beta. This finding contributes to growing awareness that type II interferon may have greater immunoregulatory potential than type I interferons.
Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans.
Authors: Kemp D J , Coppel R L , Cowman A F , Saint R B , Brown G V , Anders R F
Published: May 2006

Many proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man. As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P. falciparum cDNA sequences, cloned in Escherichia coli. The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11. About 5% of the resulting clones expressed P. falciparum sequences as polypeptides fused to beta-galactosidase. We have identified many clones that express P. falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P. falciparum growth in vitro. The antigen-positive clones contain P. falciparum cDNA sequences, as determined by hybridization. Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P. falciparum. The cloned P. falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules.
Quantitative autoradiographic localization of [3H]imipramine binding sites in the brain of the rat: relationship to ascending 5-hydroxytryptamine neuron systems.
Authors: Fuxe K , Calza L , Benfenati F , Zini I , Agnati L F
Published: May 2006

Quantitative autoradiography shows that there is a close relationship between [3H]imipramine binding sites and the distribution of 5-hydroxytryptamine (5-HT) neurons in the rat brain. High labeling is observed in the midbrain raphe nuclei, the areas of the dopamine cell groups of the substantia nigra and of the ventral tegmental area of Tsai, the ventral amygdaloid nucleus, the midline thalamic area, and parts of the hypothalmus. Thus, antidepressant drugs that have high affinity for [3H]imipramine binding sites can exert an influence at the 5-HT cell body as well as at the 5-HT nerve terminal level. The present results underline the possibility that the 5-HT and dopamine hypotheses for the mechanism of action of antidepressant drugs are not mutually exclusive, because both 5-HT and dopamine neurons can be regulated by large numbers of [3H]imipramine binding sites.
Acoustic imprinting leads to differential 2-deoxy-D-glucose uptake in the chick forebrain.
Authors: Maier V , Scheich H
Published: May 2006

This report describes experiments in which successful acoustic imprinting correlates with differential uptake of D-2-deoxy[14C]glucose in particular forebrain areas that are not considered primarily auditory. Newly hatched guinea chicks (Numida meleagris meleagris) were imprinted by playing 1.8-kHz or 2.5-kHz tone bursts for prolonged periods. Those chicks were considered to be imprinted who approached the imprinting stimulus (emitted from a loudspeaker) and preferred it over a new stimulus in a simultaneous discrimination test. In the 2-deoxy-D-glucose experiment all chicks, imprinted and naive, were exposed to 1.8-kHz tone bursts for 1 hr. As shown by the autoradiographic analysis of the brains, neurons in the 1.8-kHz isofrequency plane of the auditory "cortex" (field L) were activated in all chicks, whether imprinted or not. However, in the most rostral forebrain striking differences were found. Imprinted chicks showed an increased 2-deoxy-D-glucose uptake in three areas, as compared to naive chicks: (i) the lateral neostriatum and hyperstriatum ventrale, (ii) a medial magnocellular field (medial neostriatum/hyperstriatum ventrale), and (iii) the most dorsal layers of the hyperstriatum. Based on these findings we conclude that these areas are involved in the processing of auditory stimuli once they have become meaningful by experience.
Kinetic analysis of the interaction of human tissue kallikrein with single-chain human high and low molecular weight kininogens.
Authors: Maier M , Austen K F , Spragg J
Published: May 2006

Human low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK) have been purified to apparent homogeneity as intact, single-chain molecules. When they interacted with homologous urinary kallikrein, 0.9 mol of kinin per mol of substrate was released from LMWK and 0.7 mol of kinin per mol of substrate was released from HMWK. These functionally and structurally intact substrates have been used to obtain the kinetic constants for kinin release by purified human tissue kallikreins. With human urinary kallikrein, apparent second-order rate constants (kcat/Km) of 1.46 X 105, 8.6 X 104, and 5.08 X 104 M-1.S-1 were obtained with LMWK, HMWK, and alpha-N-p-tosyl-L-arginine methyl ester (TAMe), respectively; with human pancreatic kallikrein, values of 8.7 X 103 and 7.3 X 104 M-1.S-1 were obtained with HMWK and TAMe. These values, which are comparable to those obtained for other enzyme-protein substrate interactions, indicate that LMWK is only slightly preferred to interactions, indicate that LMWK is only slightly preferred to HMWK as the natural substrate for urinary kallikrein and that HMWK as the natural substrate for urinary kallikrein and that HMWK is a somewhat better substrate for urinary kallikrein than for pancreatic kallikrein. Although the data obtained have been shown by NaDodSO4/polyacrylamide gel electrophoresis to reflect cleavage of the substrate at two points, the linear Line-weaver-Burk plots suggest that one cleavage is rate limiting. Because the plasma concentrations of both LMWK and HMWK are approximately 1/10th the Km values obtained, substrate concentration may also play a role in determining the rate at which tissue kallikreins release kinins from kininogen substrates either in the circulation or extravascularly.
Receptor aggregation induced by antilutropin receptor antibody and biological response in rat testis Leydig cells.
Authors: Podestá E J , Solano A R , Attar R , Sánchez M L , Molina y Vedia L
Published: May 2006

Antibodies against the lutropin receptor have been obtained by the monoclonal antibody technique. Mice were immunized with luteal membrane from ovaries from pseudopregnant rats, containing high lutropin receptor concentration. Hybridoma cells were obtained by fusing mouse myeloma cells with spleen cells from the immunized animal. Five clones were produced that secreted monoclonal antibodies that specifically inhibited lutropin binding to its receptor in a competitive fashion. Antibodies from three clones were capable of blocking biological response to lutropin (e.g., testosterone production by isolated rat Leydig cells). Antibodies secreted by two other clones, however, were capable of acting as Leydig cell stimulators. Immunofluorescence studies demonstrated the presence of receptor capping which may be associated with receptor-mediated testosterone production. Antagonist antibodies could be transformed into agonist by the addition of a second crosslinking anti-mouse IgG. The discovery of agonist antibodies against the receptor molecule proves that the biological information of the lutropin-receptor complex resides in the receptor and not in the hormone.
A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man.
Authors: Nussbaum R L , Crowder W E , Nyhan W L , Caskey C T
Published: May 2006

Using cloned cDNA sequences of murine and human hypoxanthine phosphoribosyltransferase (HPRT: IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), we have identified and characterized a three-allele restriction-fragment-length polymorphism for the restriction endonuclease BamHI at the human HPRT locus. The alleles are expressed phenotypically on Southern blots as three distinct pairs of fragments that hybridize to HPRT cDNA: (i) a 22-kilobase (kb)/25-kb pair, (ii) a 12-kb/25-kb pair, and (iii) a 22-kb/18-kb pair. In addition to fragments from the HPRT locus, sequences recognized by both HPRT cDNA probes are also present on at least two autosomes in the human genome. Allele frequencies in an unselected Caucasian population are 0.77 for the 22-kb/25-kb allele. 0.16 for the 12-kb/25-kb allele, and 0.07 for the 22-kb/18-kb allele, resulting in an average heterozygosity of 38% in females in this population. This polymorphism should facilitate gene mapping by linkage in this region of the human X chromosome.
Antigenically distinct subpopulations of myeloid progenitor cells (CFU-GM) in human peripheral blood and marrow.
Authors: Ferrero D , Broxmeyer H E , Pagliardi G L , Venuta S , Lange B , Pessano S , Rovera G
Published: May 2006

Two types of progenitor cells of the human granulocytic and monocytic lineages (CFU-GM) can be distinguished by using mouse monoclonal antibodies against human hemopoietic cells. Type 1 CFU-GM contribute all of the peripheral blood CFU-GM as well as a small fraction of bone marrow CFU-GM and express surface antigens recognized by "anti-lymphomonocytic" monoclonal antibodies S3-13 and S17-25 but not the antigens recognized by R1B19 and WGHS-29-1 (two monoclonal antibodies that react with all the cells of the granulocytic lineage). Type 2 CFU-GM are present only in the marrow and react with S3-13, R1B19, and WGHS-29-1. Partial reactivity with S17-25 was observed only in the complement-dependent cytotoxicity test. In vitro culture of type 1 CFU-GM in liquid medium in the presence of granulocyte-macrophage colony-stimulatory factor (GM-CSF) generates colony-forming cells that have the surface phenotype of type 2 CFU-GM. This finding supports the idea of two different stages of maturation of myelomonocytic progenitor cells represented by type 1 and type 2 CFU-GM.
Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli.
Authors: Taylor M A , McIntosh M A , Robbins J , Wise K S
Published: May 2006

A library of cloned Mycoplasma hyorhinis genomic sequences was constructed by incorporation of EcoRI digestion fragments of mycoplasma DNA into the lambda Charon 4A bacteriophage vector. Immunological screening of recombinant phage plaques identified clones containing genes encoding mycoplasma antigenic structures expressed in an Escherichia coli host. Two such recombinant phage isolates, lambda Ch4A-MhrG1 and lambda Ch4A-MhrG28, were defined and found to contain distinct genomic sequences by analysis of restriction endonuclease fragments. Inoculation of mice with recombinant gene products from lambda Ch4A-MhrG1 yielded antiserum selectively recognizing a Mr 29,500 trypsin-sensitive mycoplasma constituent. This established a means for producing selected immunogenic mycoplasma component in a bacterial host. The cloned genomic sequences of M. hyorhinis encoding expressed mycoplasma antigens represent molecular probes that can be characterized both by specific DNA sequences and by the antigenic structure of corresponding gene products. These genomic fragments define initial physical markers of the M. hyorhinis genome and may be useful in assessing antigenic and molecular genetic relationships within the genus Mycoplasma and among other members of the class Mollicutes.
Protease inhibitors implicate metalloendoprotease in synaptic transmission at the mammalian neuromuscular junction.
Authors: Baxter D A , Johnston D , Strittmatter W J
Published: May 2006

Metalloendoproteases have been implicated in the calcium-dependent exocytosis of histamine from mast cells and in the calcium-dependent fusion of myoblasts. Because metalloendoproteases have also been identified in nervous tissue, we investigated the possibility that these proteases may be involved in neurotransmitter release at mammalian synapses. End-plate potentials were recorded intracellularly from mouse diaphragm/phrenic nerve preparations in vitro. The amplitude of the endplate potentials were reduced by as much as 90% during bath application of phosphoramidon, a specific inhibitor of metalloendoproteases, and by carbobenzoxy-dipeptide-amide synthetic substrates for metalloendoproteases. Only those synthetic dipeptides in which the amino group of the peptide bond was provided by a bulky hydrophobic amino acid, such as phenylalanine or leucine, which are substrates for metalloendoproteases, reduced synaptic transmission. Synthetic substrates in which proline or glycine provided the amino group of the peptide bond, which are not metalloendoprotease substrates, had little or no effect on the amplitude of end-plate potentials. The ability of synthetic substrates to reduce synaptic transmission was also dependent on the amino acid that provided the carboxyl group of the peptide bond, with glycine being more effective than tyrosine or serine. In addition, synthetic dipeptides with free carboxyl or amino termini, which have a low affinity for metalloendoproteases, also had little effect on synaptic transmission. The inhibition of synaptic transmission by phosphoramidon and the synthetic substrates occurred within 2 to 3 min and was completely reversible. Neither phosphoramidon nor the synthetic substrates altered the dose-response characteristics of the postsynaptic membrane to bath-applied carbachol. These results suggest that synaptic transmission requires the activity of a metalloendoprotease in the presynaptic nerve terminal and that proteolysis may be an important step during neurotransmitter exocytosis.
Thymotropism of murine leukemia virus is conferred by its long terminal repeat.
Authors: DesGroseillers L , Rassart E , Jolicoeur P
Published: May 2006

Several murine leukemia viruses (MuLV) replicate efficiently in the thymus (T+) of the mouse, whereas others are unable to replicate (T-) in this organ. To map the region of the viral genome harboring the sequences responsible for this thymotropic phenotype, we constructed viral DNA recombinants in vitro between cloned infectious viral DNAs from T- BALB/c N-tropic MuLV and from T+ BALB/c B-tropic MuLV or AKR Gross passage A MuLV. (N- and B-tropic refer to the Fv-1 host range of MuLV.) Infectious recombinant MuLVs, recovered from murine cells microinjected with these recombinant DNAs, were injected into newborn mice to test their ability to replicate in the thymus. We found that the long terminal repeat from the T+ BALB/c B-tropic or AKR Gross passage A MuLV genome was sufficient to allow replication of recombinant MuLVs in the thymus. Our sequence data suggested that the U3 tandem direct repeat was responsible for this effect. These results suggest a new role for the U3 long terminal repeat in the replication of MuLV in specific differentiated target cells.
F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein.
Authors: Kolodkin A L , Capage M A , Golub E I , Low K B
Published: May 2006

In Escherichia coli K-12 strains that carry the mutation ssb-1 in the gene for single-stranded DNA binding protein, the presence of the F sex factor partially reverses the temperature-sensitive growth phenotype caused by the mutation. The region of F (EcoRI fragment 3) responsible for this compensation has been identified and subcloned onto pBR322. A BamHI cleavage site has been found to intersect the essential coding region for this F function. By using this site, mutational blocks in the function have been constructed and used to identify a protein product (Mr approximately 22,000, slightly larger than the E. coli K-12 single-stranded DNA binding protein) which is correlated with the ssb-1-complementing activity. Labeled extracts from maxicells were used to show that this protein binds tightly to single-stranded DNA. The gene on F that codes for this protein is denoted ssf and is located at approximately 55.2 kilobases on the standard map of F, in the region transferred very early during bacterial conjugation.
cDNA clone encoding a complete rabbit immunoglobulin kappa light chain of b4 allotype.
Authors: Dreher K L , Emorine L , Kindt T J , Max E E
Published: May 2006

We have cloned a cDNA encoding a kappa light chain of the b4 allotype. The clone, pB4D5, was derived from the allotype-specific mRNA of a rabbit-mouse hybridoma 12F2, which secretes a monoclonal rabbit b4 kappa chain. Nucleotide sequence analysis of the entire pB4D5 insert reveals complete variable (V) and constant (C) regions and portions of the signal peptide and 3' untranslated region. The translated pB4D5 region amino acid sequence matches the NH2-terminal sequence of the kappa chain secreted by 12F2 and is similar, though not identical, to several other b4 kappa sequences. A V region-specific probe isolated from pB4D5 hybridizes to more than 25 bands on a genomic Southern blot of b4 rabbit DNA and yields a similar pattern with DNA from other allotypes. Sequence comparisons between pB4D5, a cloned germ-line precursor of the pB4D5 joining (J) region, and published b4 amino acid sequences reveal a peculiar feature of rabbit kappa V-J recombination in which this system resembles mouse and human heavy chain recombination more than any known kappa system.
Suspension culture of human mast cells/basophils from umbilical cord blood mononuclear cells.
Authors: Ogawa M , Nakahata T , Leary A G , Sterk A R , Ishizaka K , Ishizaka T
Published: May 2006

Selective growth of human mast cells/basophils was obtained in suspension cultures of mononuclear cells from umbilical cord blood. A fraction of culture supernatant of phytohemagglutinin-stimulated T cells, which lacked interleukin 2, was required for the selective growth of mast cells. When the mononuclear cells were cultured for 2-4 wk in the presence of the fraction, 50-90% of the total cells in the cultures contained metachromatic granules. Under the optimal culture conditions, the number of mast cells/basophils recovered from the cultures was 30-60% of the number of mononuclear cells plated. Cultured mast cells/basophils bear 1.2-3.83 X 10(5) IgE receptors per cell and contained 0.48-1.6 micrograms of histamine per 10(6) cells. The average forward rate constant, k1, and dissociation constant, k-1, for the binding of human IgE to IgE receptors on the cells were 1.9 X 10(5) M-1 sec-1 and 6.9 X 10(-5) sec-1, respectively (average equilibrium constant = 2.75 X 10(9) M-1). Specific binding of human IgE with high affinity indicates that the cells recovered in the suspension culture are human mast cells/basophils. Cultured cells sensitized with human IgE released a substantial amount of histamine upon exposure to anti-IgE. The results indicate that human mast cells/basophils obtained in the culture are functionally mature.
Stimulation of mucosal mast cell growth in normal and nude rat bone marrow cultures.
Authors: Haig D M , McMenamin C , Gunneberg C , Woodbury R , Jarrett E E
Published: May 2006

Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.
Inhibition of a major NAD(P)-linked oxidoreductase from rat liver cytosol by steroidal and nonsteroidal anti-inflammatory agents and by prostaglandins.
Authors: Penning T M , Talalay P
Published: May 2006

A NAD(P)-linked 3 alpha-hydroxysteroid dehydrogenase [3 alpha-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.50], purified to homogeneity from male rat liver cytosol, accounts for most of the oxidative activity for 3 alpha-hydroxysteroids and for benzenedihydrodiol (trans-1,2-dihydroxy-3,5-cyclohexadiene) of this tissue. This enzyme, which also promotes the reduction of quinones and certain aromatic aldehydes and ketones, is powerfully inhibited by the major types of nonsteroidal and steroidal anti-inflammatory drugs. The IC50 values for indomethacin and for betamethasone are in the low micromolar range. The rank order of the inhibitory potencies of a series of these agents paralleled closely that reported for the inhibition of cyclooxygenase by nonsteroidal anti-inflammatory agents or the indirect inhibition of phospholipase A2 by anti-inflammatory steroids. A good correlation is found also between the logarithm of the concentrations of these drugs required to inhibit 3 alpha-hydroxysteroid dehydrogenase and the human anti-inflammatory dose. The suggestion that this enzyme may play a role in mediating inflammation is further strengthened by the observation that the dehydrogenase binds arachidonic acid and is also potentially inhibited by certain prostaglandins. This enzyme may be of use in the evaluation of anti-inflammatory activity.
Free jet excitation and emission spectra of diphenylbutadiene
Authors: Heimbrook Lou Ann , Kohler Bryan E. , Spiglanin Thomas A.
Published: May 2006

Highly resolved emission and one-photon fluorescence excitation spectra for 1,4-diphenyl-1,3-butadiene seeded in a supersonic expansion of helium have been measured. The spectra show a long-lived (52.8 nsec for excitation at the 0-0) state at 29,652.5 cm
-1
, approximately 1,150 cm
-1
below the well-characterized

1
B
u

state, which is assigned as

1
A
g

—i.e., we have directly observed a polyene

1
A
g

state in the gas phase. Emission spectra and decay times for the

1
A
g

state were measured at a number of different excitation energies. These data clarify the ordering of excited singlet states and the photophysical behavior of diphenylbutadiene.
Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.
Authors: Ruta M , Bestwick R , Machida C , Kabat D
Published: May 2006

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.
A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT.
Authors: Miller A D , Jolly D J , Friedmann T , Verma I M
Published: May 2006

A cDNA corresponding to the human gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been ligated into murine retroviral vectors such that it is under the transcriptional control of viral long terminal repeats. Transfection of HPRT- cells followed by superinfection with various helper viruses has led to the rescue of chimeric virus capable of transmitting the HPRT+ phenotype to HPRT- rodent or human cells. These genetically transformed cells contain authentic human HPRT at levels similar to normal HPRT+ cells.
Molecular basis of length polymorphism in the human zeta-globin gene complex.
Authors: Goodbourn S E , Higgs D R , Clegg J B , Weatherall D J
Published: May 2006

The length polymorphism between the human zeta-globin gene and its pseudogene is caused by an allele-specific variation in the copy number of a tandemly repeating 36-base-pair sequence. This sequence is related to a tandemly repeated 14-base-pair sequence in the 5' flanking region of the human insulin gene, which is known to cause length polymorphism, and to a repetitive sequence in intervening sequence (IVS) 1 of the pseudo-zeta-globin gene. Evidence is presented that the latter is also of variable length, probably because of differences in the copy number of the tandem repeat. The homology between the three length polymorphisms may be an indication of the presence of a more widespread group of related sequences in the human genome, which might be useful for generalized linkage studies.
Long-term growth of human B cells and their use in a microassay for B-cell growth factor.
Authors: Maizel A L , Morgan J W , Mehta S R , Kouttab N M , Bator J M , Sahasrabuddhe C G
Published: May 2006

Normal human B lymphocytes, prepared from peripheral venous blood, have been stimulated with intact anti-IgM (mu chain specific) bound to an insoluble matrix. The activation event, in a subfraction of human B cells, was associated with subsequent receptivity to the mitogenic effects of exogenously added B-cell growth factor. The ability of the cell population to specifically absorb the B-cell growth factor was dependent upon the time of stimulation with the anti-IgM. Continuous replenishment of the growth factor resulted in the ability to maintain long-term growth-factor-dependent human B-cell populations. These cultured B lymphocytes were shown to specifically absorb the B-cell growth factor, suggesting the presence of membrane receptors for it. The cultured B lymphocytes were routinely maintained in logarithmic-phase growth, in the presence of growth factor, with a population doubling time of 36 hr. These cultured B cells have been utilized in a microassay for the assessment of B-cell growth factor activity that is accurate, sensitive, and precise.
Ontogenic development of contrasuppression.
Authors: Skowron-Cendrzak A , Rybczynska Z , Gershon R K , Ptak W
Published: May 2006

Using a xenogeneic graft-versus-host assay system, we have been able to document the sequential appearance of certain regulatory cells in newborn mice. Before birth, mouse spleen cells exhibit potent suppression that cannot be ameliorated by contrasuppressor cells. On the day of birth, the spleens contain equally potent suppressor cells, but these cells now can be inhibited totally by contrasuppressor cells. Between days 1 and 2 after birth, suppression, as picked up by our system, has disappeared. However, it can be found hiding behind contrasuppressor cells, as elimination of the latter cells with appropriate antisera reveals cells with the same suppressive potency as spleen cells taken 1 or 2 days earlier. Further, if the thymus is removed on the day of birth or 1 day later, the suppressor-obscuring cells do not appear. Thus, there appears to be an inherent ontogenic schedule that the thymus follows, which determines the time when different regulatory cells will emerge from it.
Low density lipoprotein degradation by mononuclear cells from normal and dyslipoproteinemic subjects.
Authors: Lees A M , Lees R S
Published: May 2006

Three major characteristics of cell surface low density lipoprotein (LDL) receptor activity in fibroblasts or lymphocytes are high-affinity LDL binding or degradation, specificity for LDL, and "inducibility"--that is, the ability to increase when cells are cultured in the absence of lipoproteins. Cells from patients with receptor-negative homozygous familial hypercholesterolemia (FH) have been reported to express none of these characteristics, and the patients are thought to have a genetic absence of LDL receptors. We found that, although induced receptor-negative lymphocytes degraded less LDL than did normal lymphocytes, the curves for LDL degradation versus LDL concentration were biphasic, with greater concentration-dependence at LDL concentrations less than 60 micrograms/ml, indicating high-affinity LDL degradation. The percentage of specific LDL degradation by induced receptor-negative lymphocytes was two-thirds of normal with LDL at 10 micrograms/ml and increased to normal at 50 micrograms/ml, an LDL concentration still within the range of high-affinity degradation. Receptor-negative lymphocytes could be induced by incubation in the absence of lipoproteins to degrade twice as much LDL as they did when freshly isolated. Freshly isolated cells from abetalipoproteinemic patients and one receptor-negative patient degraded as much LDL as did fresh normal cells. The findings indicate that receptor-negative lymphocytes have a mechanism for facilitated uptake of LDL that resembles that of normal lymphocytes, although it is not as efficient.
Human platelets contain phospholipase C that hydrolyzes polyphosphoinositides.
Authors: Rittenhouse S E
Published: May 2006

Stimulated human platelets are known to undergo marked and rapid changes in phosphoinositide metabolism consistent with the activation of phospholipase C. Such changes may promote a Ca2+ flux after platelets are exposed to agonists. I have examined this enzymatic activity by using disrupted platelets. When human platelets are sonicated and then incubated with phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) or phosphatidylinositol 4-monophosphate (PtdIns4P) in the presence of Ca2+ and deoxycholate, marked hydrolysis of these substrates occurs. Characterization of the hydrolysis products by anion exchange and thin-layer chromatography indicates that the bulk of this activity is enzymatic and attributable to phospholipase C. In the absence of Ca2+ or deoxycholate, only phosphomonoesterase activity is observed. I partially purified the soluble phospholipase C on DEAE-cellulose in order to minimize phosphomonoesterase activity. Fractions eluting at low salt concentrations contain the highest phospholipase C activity with respect to PtdIns4,5P2 and PtdIns4P and the lowest phosphomonoesterase activity. The enzyme(s) in these fractions is (are) maximally active in the presence of 0.1 mM Ca2+ and deoxycholate (1 mg/ml) and display(s) substrate affinities in the order PtdIns greater than PtdIns4P greater than PtdIns4,5P2 and maximum rates in the order PtdIns4P greater than PtdIns4,5P2 greater than PtdIns. This order of substrate preference appears to differ from that observed for physiologically stimulated cells. Possible reasons for such a discrepancy are discussed.
Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes.
Authors: Barrantes F J , Mieskes G , Wallimann T
Published: May 2006

The nonreceptor, peripheral v proteins (Mr 43,000 proteins) are conspicuous components of the acetylcholine receptor-rich membranes and the Torpedo electrocyte, so far devoid of any known enzymatic function. Creatine kinase (adenosine 5'-triphosphate:creatine N-phosphotransferase, EC 2.7.3.2) is identified in distinct polypeptides belonging to the family of v proteins. Embryonic (70- to 90-mm embryos), neonatal, and adult electric organs of Torpedo marmorata contain two isoenzymes of creatine kinase: the BB (brain) and the MM (muscle) forms. The proportion of the two isoenzymes does not appear to change in the course of ontogenic and postnatal development. Only the BB isoenzyme appears to be associated with the acetylcholine-rich membranes in adult Torpedo. The creatine kinase can be purified to homogeneity by chromatographic procedures that exploit the richness in free sulfhydryl groups of the enzyme. Specific activities of 150 units/mg are obtained from electric tissue. The enzyme subunits identified by two-dimensional gel electrophoresis and immunoblotting techniques have pI values in the 6.0-6.5 region and apparent molecular weights in the 40,000-43,000 range, the latter values depending on redox conditions.
Multiple steady states and oscillatory behavior of a compartmentalized phosphofructokinase system.
Authors: Hervagault J F , Duban M C , Kernevez J P , Thomas D
Published: May 2006

This paper discusses interacting diffusion and reaction in an open enzyme system. The enzyme, rabbit muscle phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), is inhibited strongly by excess of the substrate ATP. Metabolites diffuse through an inert membrane separating the enzyme from the bulk reacting medium. We demonstrate that such a simple system is able to account for the existence of both oscillatory behavior (limit cycle) and multiple steady states (hysteresis) as well as for the sudden transitions between stable and periodic behaviors. Experimental evidence for time oscillations is given.