bluuebunny/crossref_metadata_2025 huggingface dataset:
Arthritis induced by a T-lymphocyte clone that responds to Mycobacterium tuberculosis and to cartilage proteoglycans.
Authors: van Eden W , Holoshitz J , Nevo Z , Frenkel A , Klajman A , Cohen I R
Published: May 2006

Adjuvant arthritis characterized by chronic inflammation of the joints of rats is induced by immunization to Mycobacterium tuberculosis. To learn how autoimmune arthritis may be caused by a microbial antigen, we isolated a T-lymphocyte clone specific for M. tuberculosis antigens that was strongly arthritogenic. We now report that the clone recognized, in addition to M. tuberculosis antigens, antigens present in human synovial fluid, medium of chondrocyte cultures, and proteoglycans purified from cartilage. These observations indicate that the target antigen for the arthritogenic clone resides in the proteoglycan component of cartilage. As this arthritogenic clone shows specificity for both a M. tuberculosis antigen and a cartilage constituent we conclude that disease is probably caused by antigenic cross-reactivity. Thus, an autoimmune disease may be triggered by structural mimicry between antigens in the environment and self-antigens in the individual.
A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein.
Authors: Coppel R L , Favaloro J M , Crewther P E , Burkot T R , Bianco A E , Stahl H D , Kemp D J , Anders R F , Brown G V
Published: May 2006

A cDNA clone expressing a Plasmodium falciparum blood-stage antigen in Escherichia coli was identified by colony immunoassay using immune human sera. Antibodies affinity-purified on extracts of this clone reacted with both asexual blood stages and sporozoites of P. falciparum, recognizing a Mr23,000 protein in the blood stages. The nucleotide sequence of the cDNA revealed a signal peptide and an internal hydrophobic sequence typical of transmembrane anchor sequences. Located 3' to the putative anchor are two tetramers, Asn-Ala-Asn-Pro and Asn-Ala-Asp-Pro, which are closely related to the repeats of the circumsporozoite protein of P. falciparum. The blood stage protein is conserved amongst several isolates of P. falciparum, and antibodies against it are common in the sera of individuals living in the area where the parasite is endemic.
In vivo separation of two classes of T cells as determined by negative selection after the injection of UV-treated allogeneic lymphoid cells.
Authors: Bellgrau D , Lagarde A C
Published: May 2006

Negative selection occurred when gamma-irradiated lymphoid cells of rat strain PVG were injected intravenously into strain Fischer rats, which differ from PVG rats at the major histocompatibility complex. By 48 hr after injection, thoracic duct lymphocytes (TDL) from these animals failed to proliferate or generate cytotoxic T lymphocytes (CTL) in vitro in response to gamma-irradiated PVG lymphoid cells. Responses to cells of a third rat strain were unaffected. By about 6 days after antigen injection, positive selection had occurred, as shown by enhanced responses to gamma-irradiated PVG cells in the same assays. Ultraviolet-irradiated strain PVG populations were tested in the same way. They differed from gamma-irradiated PVG cells in that they failed to induce proliferation or stimulate CTL in vitro. TDL from Fischer rats injected with UV-irradiated PVG cells failed to proliferate or generate CTL in response to gamma-irradiated PVG cells in vitro. The CTL response of TDL from rats injected with UV-irradiated cells was restored by the addition of supernatant factor(s) from concanavalin A-stimulated lymphoid cells (ConA SnF). Therefore, CTL precursors did not undergo negative selection after injection of UV-treated cells. There was also no detectable positive selection 6 days after injection of UV-treated cells. These experiments show that it is possible to separate two populations of T cells in vivo by the injection of UV-irradiated allogeneic lymphoid cells. One population incorporates [3H]thymidine early in an allogeneic response in vitro, the other expresses cytotoxic activity only after the addition of ConA SnF in vitro. The first population can replace the requirement for ConA SnF by the CTL population.
Constants and pseudo-constants of the Kadomtsev-Petviashvili equation
Authors: Case K. M.
Published: May 2006

Elucidating earlier work, it is shown that the Kadomtsev-Petviashvili equation has
n
+ 2 constants for all
n
≥ 0. It also has a pseudo-constant from which the constants can be obtained by differentiation with respect to time. The pseudo-constant can be obtained from a basis functional
J

n

(
n
+2)

= -1/18 [unk]
y

n
+2

q
by taking repeated Poisson brackets with the Hamiltonian.
Exuberant restriction fragment length polymorphism associated with the DQ alpha-chain gene and the DX alpha-chain gene.
Authors: Le Gall I , Marcadet A , Font M P , Auffray C , Strominger J , Lalouel J M , Dausset J , Cohen D
Published: May 2006

Cellular DNAs from individuals of 23 families were digested with five restriction endonucleases (Pvu II, EcoRI, HindIII, BamHI, and EcoRV) and then probed with a DX alpha-chain gene probe. Seventeen allogenotopes were observed, each of which could be assigned to a serologically defined haplotype by noting its segregation in families. Six sets of allogenotopes forming allelic series were noted. In comparison with restriction maps of the DQ alpha and the DX alpha regions, each of these series has been assigned to the DQ alpha or the DX alpha locus. Allogenotopes of the four DQ alpha series constitute three clusters correlating with the supertypic groups of class II histocompatibility antigens DQw1 (DR1, DR2, and DRw6), DRw53 (DR4, DR7, and DR9), and DR3 plus DR5 plus DR8. These 13 DQ alpha fragments constitute 22 different patterns. The two DX alpha series constitute two clusters, one of which is not found to be correlated strongly with DR specificities, whereas the other is correlated loosely (r = 0.45) with DR5 and DR7. This absence of strong linkage disequilibrium between the DX alpha series and the DR series contrasts with the DQ alpha series and suggests a recombination point between DQ alpha and DX alpha loci.
The Escherichia coli supX locus is topA, the structural gene for DNA topoisomerase I.
Authors: Margolin P , Zumstein L , Sternglanz R , Wang J C
Published: May 2006

Mutations in the supX locus, which result in the absence of DNA topoisomerase I enzyme activity in both Salmonella typhimurium and Escherichia coli, are all selected as suppressors of the leu-500 promoter mutation in S. typhimurium. To determine whether the supX locus is the structural gene topA for the DNA topoisomerase I enzyme or is a positive-acting regulator/activator gene for a nearby topA structural gene, nonsense mutations were selected in the E. coli supX gene carried on an F' episome in S. typhimurium cells. The cysB-topA region of the episomes with nonsense-mutant supX alleles were then cloned onto plasmid pBR322 and transformed into E. coli cells lacking a chromosomal supX gene. Three such E. coli strains, each carrying cloned DNA from episomes with different nonsense-mutant supX alleles, all lacked DNA topoisomerase I activity but expressed antigenic determinants specific to the enzyme; control cells lacked both enzyme activity and antigenic determinants. Maxicell studies of plasmid-coded proteins demonstrated the absence of the DNA topoisomerase I protein (100 kDa) in the three strains but the appearance of a new smaller peptide in each (36, 47, and 64 kDa). These new peptides must represent fragments of the enzyme resulting from translation termination at the supX nonsense codons and confirm the interpretation that the supX gene is topA, the structural gene for DNA topoisomerase I.
Differential expression of the mouse cholecystokinin gene during brain and gut development.
Authors: Friedman J , Schneider B S , Powell D
Published: May 2006

Cholecystokinin (CCK) is a neuropeptide found in brain and intestine. In this report, we have isolated a cDNA clone that encodes CCK from a mouse brain cDNA library. This cDNA clone has extensive homology to CCK precursors that have been sequenced previously. Southern blots of genomic DNA probed with this cDNA clone revealed single bands for each of eight different restriction enzymes, all of which could be accounted for by a single genomic clone, suggesting that the CCK gene is present as a single-copy gene in mice. RNA blots, primer extensions, and S1 nuclease protection assays have suggested that the same RNA start site is utilized in brain and in gut. Finally, we have shown, by using RNA blots and a radioimmunoassay specific for CCK, that CCK is expressed at maximum adult levels in intestine at birth but that adult concentrations of CCK and its mRNA are not reached in brain until much later in development.
Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity.
Authors: Billingsley M L , Kincaid R L , Lovenberg W
Published: May 2006

Calcineurin, a calmodulin-stimulated protein phosphatase, was a substrate for purified bovine brain protein carboxyl O-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) and incorporated up to 2 mol of CH3 per mol of calcineurin. Carboxyl methylation was dependent on the concentrations of S-adenosyl-L-[methyl-3H]methionine and was prevented by addition of the carboxyl methylation inhibitor S-adenosylhomocysteine. The stoichiometry of methyl group incorporation was related to the ratio of methyltransferase/calcineurin. The rate of spontaneous hydrolysis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for trichloracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO4 (pH 2.4) confirmed that the A subunit of calcineurin (Mr = 61,000) was the primary site of carboxyl methylation with little, if any, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (approximately 1-2 mol of CH3 per mol of protein) was assayed for p-nitrophenyl phosphatase activity at pH 6.5, a marked inhibition of calmodulin-stimulated activity was observed while there was little effect on Mn2+-stimulated phosphatase activity. Thus, calcineurin appears to be an excellent substrate for protein carboxyl O-methylation and this modification, which impairs calmodulin stimulation of phosphatase activity, may be of functional significance.
Amino acid sequence of the UP1 calf thymus helix-destabilizing protein and its homology to an analogous protein from mouse myeloma.
Authors: Williams K R , Stone K L , LoPresti M B , Merrill B M , Planck S R
Published: May 2006

A complete amino acid sequence has been determined for the UP1 single-stranded DNA binding protein from calf thymus that was first described by G. Herrick and B. M. Alberts [(1976) J. Biol. Chem. 251, 2124-2132]. Peptides required to establish the UP1 sequence were isolated by reversed-phase HPLC of digests produced by endoproteinase Lys-C, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage of UP1. The purified peptides were coupled to aminopolystyrene prior to solid-phase sequencing. UP1 contains 195 amino acids and has a molecular weight of 22,162. UP1 has a blocked NH2 terminus and contains a single NG,NG-dimethylarginine residue near its COOH terminus. Gas-phase sequencing of tryptic peptides derived from an analogous protein from mouse myeloma cells [Planck, S. R. & Wilson, S. H. (1980) J. Biol. Chem. 255, 11547-11556] revealed that this mouse helix-destabilizing protein shares a high degree of sequence homology with UP1. Of the 59 amino acids in the mouse protein that have so far been found to be homologous with UP1, 48 correspond exactly to sequences found in UP1. Most of the 11 differences that have been found between the sequences of these two proteins are conservative in nature, involving primarily the interchange of chemically similar amino acids. One 9-residue mouse sequence that is not obviously homologous to UP1 may be a result of the larger size of the mouse myeloma protein as compared to UP1. Since none of the UP1 or mouse myeloma helix-destabilizing protein sequence appears to be homologous to that of any previously sequenced protein, we presume that these two proteins represent a distinct class of single-stranded nucleic acid binding proteins that probably play a role in metabolism of single-stranded RNA or DNA in vivo.
Isolation of tumor-secreted products from human carcinoma cells maintained in a defined protein-free medium.
Authors: Alderman E M , Lobb R R , Fett J W
Published: May 2006

A protein-free synthetic cell-growth medium has been defined that permits long-term survival (greater than 120 days) of an established human colon tumor cell line, HT-29. Viability is dependent upon both the concentration of L-glutamine in the medium and the cell density at the time of initial transfer into it. Cell proliferation is minimal, thus obviating the necessity for subculturing. HT-29 adenocarcinoma cells maintained in large-scale culture with this medium continue to secrete the established colon tumor marker carcinoembryonic antigen as well as growth factors and lysozyme. These and, potentially, other important tumor-derived products can therefore be generated continuously in such cultures so that they can be isolated from a conditioned medium free of contaminating serum and protein supplements.
Conservation of nodulation genes between
Rhizobium meliloti
and a slow-growing
Rhizobium
strain that nodulates a nonlegume host
Authors: Marvel Deborah J. , Kuldau Gretchen , Hirsch Ann , Richards Eric , Torrey John G. , Ausubel Frederick M.
Published: May 2006

Parasponia
, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a
Rhizobium
species. The bacterial strain RP501 is a slow-growing strain of
Rhizobium
isolated from
Parasponia
nodules. Strain RP501 also nodulates the legumes siratro (
Macroptilium atropurpureum
) and cowpea (
Vigna unguiculata
). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb)
Eco
RI fragment that complemented insertion and point mutations in three contiguous nodulation genes (
nodABC
) of
Rhizobium meliloti
, the endosymbiont of alfalfa (
Medicago sativa
). The complemented
R. meliloti nod
mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or
Parasponia
. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the
R. meliloti nodABC
genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb
Eco
RI fragment prevented complementation of
R. meliloti nodABC
mutations.
Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus.
Authors: Ranelli D M , Jones C L , Johns M B , Mussey G J , Khan S A
Published: May 2006

We have cloned the Staphylococcus aureus entB gene in Escherichia coli, using pBR322 as the vector plasmid; however, no detectable staphylococcal enterotoxin B (SEB) was produced by the E. coli clones. When the entB gene was placed downstream from the strong lambda phage promoter, PR, SEB was synthesized at readily detectable levels in E. coli. Interestingly, mature SEB was almost exclusively present in the cytoplasmic fraction. The SEB precursor was found associated with the cell membrane. The entB gene was introduced back into S. aureus, and the clones were shown to produce SEB. The entB gene has been located to a 2.1-kilobase-pair region. Maxam-Gilbert sequencing of a part of the entB gene yielded a DNA sequence that corresponds to the known amino acid sequence of SEB. Southern hybridization experiments showed that the entB gene was present on identical restriction fragments in the chromosomes of SEB-producer strains. The entB gene is absent from SEB-nonproducer strains.
Nucleotide sequence of an unusual regionally expressed silkmoth chorion RNA: predicted primary and secondary structures of an architectural protein.
Authors: Regier J C , Pacholski P
Published: May 2006

We have sequenced DNA clones corresponding to the entire coding and 5' untranslated regions and almost all of the 3' untranslated region of a silkmoth chorion RNA which is expressed largely in a subpopulation of follicular epithelial cells (aeropyle crown region). This RNA encodes the E1 protein, one of two components of the prominent "filler" that helps mold the shape of aeropyle crowns. The conceptually translated E1 sequence reveals an alternation in hydrophobic and hydrophilic stretches of amino acids that correlates with certain predictions about its secondary structure. E1 is unusual in revealing no sequence homology with other known chorion sequences and in having an unusually long 3' untranslated region. Sequence analysis of the 5' end of the E1 gene has identified an intron near the end of the signal peptide-encoding region, a feature shared with other chorion genes.
Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.
Authors: Reznick A Z , Rosenfelder L , Shpund S , Gershon D
Published: May 2006

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins.
Electronegativity and hardness as coordinates in structure stability diagrams.
Authors: Shankar S , Parr R G
Published: May 2006

With electronegativity and hardness of an atom defined as 1/2(I + A) and 1/2(I - A), respectively, where I and A are the ionization potential and electron affinity, electronegativity difference and hardness sum are proposed as coordinates in structure stability diagrams. With these coordinates a successful topological classification of the crystal structures of octet and suboctet binary compounds is obtained, and a clear delineation of the structural classes portraying chemical periodicity is found.
Phosphorylation enzymes of the propionic acid bacteria and the roles of ATP inorganic pyrophosphate, and polyphosphates.
Authors: Wood H G , Goss N H
Published: May 2006

It is shown that polyphosphates are not generated in significant amounts in the phosphoglycerate kinase reaction; polyphosphate is more effective than ATP in the formation of glucose 6-P by glucokinase, but the rate with ATP may be adequate to meet the requirements of glucose metabolism; PPi is far more effective than ATP as a phosphate donor in the formation of fructose 1,6-P2 by phosphofructokinase; PPi rather than ATP almost certainly is used in this reaction; and, aside from glucokinase and phosphofructokinase, the enzymes of phosphorylation are specific in their requirements of phosphate donors or acceptors and are present in adequate amounts to meet the requirements of glucose metabolism by the propionic acid bacteria.
Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.
Authors: Evans L H , Cloyd M W
Published: May 2006

A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses arose by recombination of the respective ecotropic MuLVs with different endogenous retrovirus sequences of NFS mice.
Specific transcription of preformed nucleoprotein complexes, containing the adenovirus major late promoter, with a chromatographic fraction containing RNA polymerase II.
Authors: Hellwig R J , Sinha S N , Niyogi S K
Published: May 2006

Incubation in a HeLa whole-cell extract converted a plasmid DNA containing the adenovirus type 2 major late promoter into ordered nucleoprotein complexes similar to those described for simian virus DNA [Sinha, S. N., Hellwig, R. J., Allison, D. P. & Niyogi, S. K. (1982) Nucleic Acids Res. 10, 5533-5552]. Purified nucleoprotein complexes containing the plasmid DNA were able to serve as template for accurate transcription in vitro. Use of such nucleoprotein complexes eliminated the need for addition of nontemplate DNA (poly[d(I-C)]) to transcription reaction mixtures with template concentrations too low to yield a detectable specific-transcription signal from "naked" DNA. Of the four fractions resulting from phosphocellulose column chromatography of the whole cell extract, only the fraction that contained the RNA polymerase II activity was needed to accurately transcribe these nucleoprotein complexes in the presence of human placental ribonuclease inhibitor. In contrast, specific transcription of naked template DNA under these conditions required at least one additional fraction containing specificity factors. These results show that the nucleoprotein complexes contain factor(s) needed for specific initiation of transcription and that these complexes may be useful in the purification and analysis of these factors.
Locating a nucleotide-binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences.
Authors: Gentry G A
Published: May 2006

Computer techniques were used to locate related segments of amino acid sequences in the thymidine kinases of vaccinia virus and of herpes simplex virus type 1 and in porcine adenylate kinase. As determined by a procedure that evaluates triply aligned sequences, the probability that the similarities among the segments described here arose by chance was no greater than 0.001. Because the sequence in porcine adenylate kinase is a nucleotide phosphate-binding site it is concluded that the segments in the vaccinia virus and herpes simplex virus thymidine kinases perform similar functions. The segments are residues 16-23 in porcine adenylate kinase, 11-19 in vaccinia virus thymidine kinase, and 56-64 in herpes simplex virus thymidine kinase.
The production of DNA strand breaks in human leukocytes by superoxide anion may involve a metabolic process.
Authors: Birnboim H C , Kanabus-Kaminska M
Published: May 2006

H2O2 is known to be capable of inducing strand-break damage in intracellular DNA, but whether O2- also can do so in the absence of H2O2 is uncertain. The difficulty in distinguishing the effects of the two is that, under physiological conditions, dismutation of O2- to H2O2 can readily occur. When human leukocytes are stimulated with phorbol 12-myristate 13-acetate (PMA), they release O2-, and within a few minutes strand breakage in intracellular DNA can be observed. We have attempted to determine whether the O2- produced is itself capable of causing DNA damage or whether H2O2 alone, or in combination with O2-, is responsible for the observed damage. Addition of catalase (up to 250 micrograms/ml) to remove H2O2 prevented no more than about 50% of the DNA damage. The majority of the remaining damage could be blocked, in a dose-dependent manner, by superoxide dismutase (SOD) or a SOD-mimetic copper complex, identifying a fraction of damage to intracellular DNA dependent upon extracellular O2-. We studied this O2(-)-specific fraction through the use of three metabolic poisons (fluoride, 2-deoxyglucose, and A23187). These agents largely blocked DNA damage, while affecting extracellular O2- levels only slightly. For comparison, H2O2-induced DNA damage was studied with glucose oxidase to generate a flux of H2O2. The first two metabolic poisons had little effect, whereas A23187 did inhibit H2O2-induced DNA damage. We conclude that O2(-)-induced damage occurs through a mechanism that differs, at least in part, from the H2O2 damage pathway and that the former may involve one or more metabolic steps.
Identification of a high molecular weight polypeptide that may be part of the circadian clockwork in
Acetabularia
Authors: Hartwig R. , Schweiger M. , Schweiger R. , Schweiger H. G.
Published: May 2006

In the chloroplast fraction of the unicellular and uninucleate green alga
Acetabularia
, we have detected a

M
r

≈230,000 protein (p230) whose synthesis exhibits a pronounced endogenous diurnal rhythm. As judged by scanning densitometry of fluorographs of NaDodSO
4
/polyacrylamide gels, the synthesis of other proteins in the same fraction was independent of the time in the cycle. The incorporation of [
35
S]methionine into p230 was completely inhibited by cycloheximide, whereas chloramphenicol had no effect. This strongly suggests that p230 is translated on 80S ribosomes. Eighthour periods of exposure to cycloheximide produced a shift in the phase of the oscillation of p230 synthesis. The results are consistent with the hypothesis that p230 is essential for expression of circadian rhythms in
Acetabularia
.
Gene localization on sorted chromosomes: definitive evidence on the relative positioning of genes participating in the mouse plasmacytoma-associated typical translocation.
Authors: Wirschubsky Z , Ingvarsson S , Carstenssen A , Wiener F , Klein G , Sümegi J
Published: May 2006

Experiments have been carried out to establish the relative position of the mouse Ig heavy chain locus and the c-myc oncogene. In mouse plasmacytoma with the typical rcpt(12;15) chromosome translocation the c-myc oncogene is juxtaposed to one of the heavy chain genes in a head-to-head orientation. Since the relative orientations of the c-myc locus and the Ig heavy chain gene cluster on the corresponding mouse chromosomes had not been settled, it was not known whether the rearranged c-myc gene is transposed to chromosome 12 or remains on chromosome 15. To decide which of the two alternatives is correct, we separated the translocation chromosomes by fluorescence-activated chromosome sorting. The separated chromosomal fractions were hybridized with myc-specific DNA probes corresponding to the first or second/third exons in a chromosome spot assay. The results presented here indicate that the c-myc gene in mouse plasmacytoma carrying the typical translocation, as in the human Burkitt lymphoma analogous translocation, transposes to the chromosome carrying the Ig heavy chain locus. These results also establish the orientations of the Ig heavy chain locus and the c-myc locus on their normal chromosomes.
Translocation of c-myc in the hereditary renal cell carcinoma associated with a t(3;8)(p14.2;q24.13) chromosomal translocation.
Authors: Drabkin H A , Bradley C , Hart I , Bleskan J , Li F P , Patterson D
Published: May 2006

A translocation between chromosomes 3 and 8, t(3;8)(p14.2;q24.13), has been reported in a family with hereditary renal cell carcinoma. Using somatic cell hybrids, we have isolated, separately, both derivative chromosomes. We find that the c-myc oncogene (8q24.1) has been translocated to the derivative 3 [der(3)]. We have not detected a rearrangement within an approximately equal to 21-kilobase region around the c-myc gene using restriction enzyme digestion and Southern blot hybridization analysis. The translocated c-myc gene should provide a probe to the chromosome 3p14 region, which appears to be important not only in renal cell carcinoma but also in small cell carcinoma of the lung. These hybrids have also been useful for the regional mapping of the Chinese hamster ovary cell Gly-B defect to 8q22.1----q24.13 and support the regional assignment of acylase I to 3p21.
cDNA of the immunoglobulin kappa chain of an Epstein-Barr virus-transformed human lymphoid cell line: partial sequence determination and bacterial expression.
Authors: Morin J W , Black A , Wu M , Beychok S
Published: May 2006

We report the isolation, nucleotide sequence determination, and bacterial expression of a partial cDNA for the immunoglobulin kappa chain from the Epstein-Barr virus-transformed human lymphoid cell line GM131. The cDNA, cloned in pBR322 by use of oligo(dG) X oligo(dC) tails, yields two Pst I fragments of 250 and 600 base pairs (bp). Various restriction enzyme fragments of the cDNA were subcloned in the vectors M13 mp10 and M13 mp11 for sequence analysis. As a result of instability of the 250-bp M13 subclones, the base sequence of the 250-bp Pst I fragment could not be determined. The 600-bp Pst I fragment contains coding sequences for part of the variable (V) region (residues 78-95) and all of the joining (J) (residues 96-108) and constant (C) regions (residues 109-212) and extends 148 bp into the 3' flanking region. Although the C- and 3'-flanking-region sequences are identical to germ-line sequences, the J-region sequence does not correspond to any of the five human germ-line J regions. The sequence is most similar to that of J4, with three base changes resulting in one silent mutation and two amino acid substitutions, at residues 103 (Lys----Tyr) and 106 (Ile---Met). The silent mutation appears to be the result of RNA splicing between the J and the C regions. The V-region sequence differs from published V-region germ-line sequences at several codons and from the more common amino acid sequences at two positions, residues 91 and 93. At these positions, histidine residues are found in place of the more common tyrosine and serine, respectively. None of the four amino acid substitutions observed for the GM131 kappa-chain are unique, suggesting that the changes, which most likely contribute to antigenic specificity, are compatible with antibody structure and function. The 600-bp Pst I fragment was subcloned in two prokaryotic expression vectors, pATH11 and pUC8. In both instances, a kappa-chain fusion protein detectable by immunoblotting was produced.
H-2-incompatible bone marrow chimeras produce donor-H-2-restricted Ly-2 suppressor T-cell factor(s).
Authors: Noguchi M , Onoé K , Ogasawara M , Iwabuchi K , Geng L , Ogasawara K , Good R A , Morikawa K
Published: May 2006

To study adaptive-differentiation phenomena of T lymphocytes, suppressor T-cell factors (TsF) produced by Ly-2+ splenic T cells from fully allogeneic mouse bone marrow chimeras were analyzed. AKR mice irradiated and reconstituted with B10 marrow cells (B10----AKR chimeras) produced an Ly-2+ TsF after hyperimmunization with sheep erythrocytes. The TsF suppressed primary antibody responses (to sheep erythrocytes) generated with spleen cells of mice of H-2b haplotype but not those of H-2k haplotype. Thus, this suppressor factor was donor-H-2-restricted. The immunoglobulin heavy chain variable region gene (Igh-V)-restricting element was not involved in this form of suppression. Similar results were obtained when TsF from B6----BALB/c and BALB/c----B6 chimeras were analyzed. The TsF from B10----AKR chimeras suppressed responses of B10.A(3R) and B10.A(5R) mice but not those of B10.A(4R). This finding showed that identity between the factor-producing cells and target spleen cells is required on the left-hand side of the E beta locus of the H-2 region and that the putative I-Jb locus is not involved in this form of suppression. The present results support the postulate that post-thymic differentiation in the presence of continued or repeated stimulation with antigen and donor-derived antigen-presenting cells generates donor-H-2-restricted T-cell clones that may predominate within the repertoire of the specific antigen being presented.
Light-activated guanosinetriphosphatase in Musca eye membranes resembles the prolonged depolarizing afterpotential in photoreceptor cells.
Authors: Blumenfeld A , Erusalimsky J , Heichal O , Selinger Z , Minke B
Published: May 2006

Measurement of light-dependent GTPase (EC 3.1.5.1) activity in a paradigm guided by electrophysiological experiments was used to examine the involvement of a guanine nucleotide binding protein in fly phototransduction. Cell-free membrane preparations of Musca eyes responded to blue light by a 10- to 20-fold increase in GTP-hydrolyzing activity. This light-dependent GTPase had a low Km for GTP (0.5 microM) and was effectively inhibited by guanosine (5'----O3)-1-thiotriphosphate and guanosine 5'-[beta-gamma-imino]triphosphate but not by adenosine 5'-[beta-gamma-imino]triphosphate and ATP. The action spectrum of GTPase activity measured with intense light resembled closely the photoequilibrium spectrum of metarhodopsin. After illumination with blue (less than 480 nm) light, which converted rhodopsin to metarhodopsin, the GTPase remained highly active for at least 60 min in the dark. Similarly, rhodopsin-to-metarhodopsin conversion in intact cells induced a prolonged excitation in the dark, known as the prolonged depolarizing afterpotential (PDA). The persistent GTPase activity (like the PDA) was suppressed to the low basal activity of the unilluminated membranes after conversion of metarhodopsin to rhodopsin with red light (greater than 570 nm), whereas during illumination with red light, some GTPase activity was maintained. The magnitude of the persistent GTPase activity in the dark, like the PDA, depended in a supralinear manner on the amount of pigment conversion. Thus, the dependence of GTPase activity of Musca membrane preparations on photopigment conversion resembles the induction and suppression of the PDA measured in intact photoreceptors of Musca. These findings indicate that a guanine nucleotide binding protein is part of the chain of events leading to both the generation of the receptor potential and the PDA.
Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen.
Authors: Piette J , Decuyper-Debergh D , Gamper H
Published: May 2006

Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively. The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli. The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA. A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts. Numerous mutant clones partially or totally deficient in beta-galactosidase were plaque-purified and amplified. The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure. All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations. The predominant mutation was a T.A----G.C transversion.
Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.
Authors: Uno I , Mitsuzawa H , Matsumoto K , Tanaka K , Oshima T , Ishikawa T
Published: May 2006

Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 produced GTP-independent adenylate cyclase and a large amount of cAMP. Production of cAMP in the transformant carrying CYR1 and either RAS2 or RAS2val19 was confirmed by staining colonies on maltose-MacConkey plates and by measuring induction of beta-galactosidase by isopropyl beta-D-thiogalactopyranoside. Mixing a crude extract from the E. coli transformant carrying CYR1 with a crude extract from cells carrying RAS2 reconstituted the GTP-dependent adenylate cyclase. Reconstitution of the GTP-dependent adenylate cyclase was observed by mixing the plasma membrane fraction of yeast CYR1 ras1 ras2 bcy1 mutant and a crude extract from the E. coli transformant carrying RAS2 or by mixing a crude extract from the E. coli transformant carrying CYR1 and the membrane fraction of yeast cyr1 RAS1 RAS2 BCY1 mutant. The data suggest that the yeast GTP-dependent adenylate cyclase consists of catalytic and regulatory subunits encoded by the CYR1 and RAS2 genes, respectively.
An antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity.
Authors: Herrera R , Petruzzelli L , Thomas N , Bramson H N , Kaiser E T , Rosen O M
Published: May 2006

Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the beta subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the beta subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the beta subunit suggests that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor.
Anti-peptide antibodies detect oncogene-related proteins in urine.
Authors: Niman H L , Thompson A M , Yu A , Markman M , Willems J J , Herwig K R , Habib N A , Wood C B , Houghten R A , Lerner R A
Published: May 2006

Antisera to a number of synthetic peptides predicted from nucleic acid sequences of oncogenes have been used to screen 483 urine samples of cancer patients, pregnant women, and normal controls for the presence of immunologically related proteins. Increased levels of oncogene-related proteins are found during neoplasia and pregnancy. The differential detection of these oncogene-related proteins indicates that panels of monoclonal antibodies may provide a convenient noninvasive means of detecting, classifying, and staging a wide variety of malignancies and may be useful in following fetal development during pregnancy.
Mechanism of action of the group A streptococcal C5a inactivator.
Authors: Wexler D E , Chenoweth D E , Cleary P P
Published: May 2006

Virulent group A streptococci have been found to express a cell-surface factor that has the capability of inactivating complement-derived chemotactic factors. To determine the mechanism of action of this factor, we examined the interaction of purified inactivator with pure C5a chemotaxin. Ligand-receptor binding studies demonstrated that streptococcal chemotactic factor inactivator (SCFI)-treated C5a expressed a greatly reduced ability to bind to receptors of polymorphonuclear leukocytes as compared with native C5a. The inactivation of C5a occurred by a nonstoichiometric and temperature-dependent process. NaDodSO4/PAGE analysis indicated that SCFI mediated a small decrease in the molecular weight of C5adesArg, and sequencing of the carboxyl terminus of inactivated C5a demonstrated that a six-residue peptide was lost. The release of discrete peptide fragments from denatured bovine serum albumin upon prolonged incubation with SCFI was indicative of endoprotease activity. Although denatured bovine serum albumin was inefficiently cleaved, native bovine serum albumin and other native proteins were highly resistant to SCFI proteolysis; this indicated that activity was specific in nature.
Driving force and nucleophilicity in S
N
2 displacements
Authors: Streitwieser Andrew
Published: May 2006

The free energies of activation for reaction of six anionic nucleophiles with methyl iodide in dimethylformamide correlate linearly with the overall heats of reaction in the gas phase. The result indicates that nucleophilicity in this S
N
2 displacement reaction is dominated by electron affinity and bond-strength effects.
A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus.
Authors: Dorsch-Häsler K , Keil G M , Weber F , Jasin M , Schaffner W , Koszinowski U H
Published: May 2006

Using the simian virus 40 "enhancer trap" approach, we have identified a transcription enhancer located just upstream of the major immediate early gene of murine cytomegalovirus. This enhancer has several striking properties. (i) Together with the enhancer of human cytomegalovirus, it is the strongest transcription enhancer found to date. (ii) It is an extremely long enhancer, spanning greater than 700 base pairs. (iii) It consists of a rather complex pattern of sequence repeats, the longest of which is 181 base pairs. Also, several types of short sequence motifs are scattered throughout the enhancer in monomeric, heterodimeric, or homodimeric (palindromic) form. These motifs have been identified to be components of other enhancers and promoters, and they are presumably binding sites for specific nuclear factors. Our analysis suggests that enhancers are composed of a modular arrangement of short conserved sequence motifs and that enhancer strength is correlated with the redundancy of these motifs.
Identification of a novel receptor in Drosophila for both epidermal growth factor and insulin.
Authors: Thompson K L , Decker S J , Rosner M R
Published: May 2006

The notable amino acid homology among mammalian growth factor receptors with tyrosine-specific protein kinase activity has led to speculation that these receptors derived from a common evolutionary precursor. We report the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100-kDa protein is also related antigenically to the mammalian receptors for EGF and possibly insulin but may not correspond to the mammalian counterpart of either receptor in Drosophila. The Drosophila protein is recognized by antisera directed against the mammalian receptor for EGF in immunoblot hybridizations. It can be affinity labeled with either 125I-labeled insulin or 125I-labeled EGF after immunoprecipitation with anti-EGF receptor antiserum. Excess unlabeled EGF or insulin will block the affinity labeling with either growth factor, suggesting that both EGF and insulin share a common binding site on the 100-kDa Drosophila receptor. This Drosophila protein, therefore, may be closely related to an evolutionary precursor of the mammalian receptors for insulin and EGF.
Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets.
Authors: Stearns M E , Jenkins D P , Tew K D
Published: May 2006

Fluorescence-microscopic studies with dansylated estramustine (DnsEM) has permitted investigation of the mechanism of estramustine (EM) uptake in live human prostatic tumor cells (DU 145). DnsEM appeared to enter cells rapidly at the peripheral cell margins. A progressive increase in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous newly created vesicles that accumulate in the cytoplasm and around the nucleus. Colony-forming assays showed EM and DnsEM to be equally cytotoxic to cultured DU 145 cells. Cellular uptake and subsequent manifestation of cytotoxicity are presumably dependent upon these vesicles. However, after uptake of DnsEM, its diffusion into the cytoplasm was observed.
Multistep transformation by defined fragments of herpes simplex virus type 2 DNA: oncogenic region and its gene product.
Authors: Hayashi Y , Iwasaka T , Smith C C , Aurelian L , Lewis G K , Ts'o P O
Published: May 2006

Diploid Syrian hamster embryo cells transfected with Bgl II C fragment of herpes simplex virus type 2 DNA acquired a neoplastic phenotype. Cultures transfected with its left-hand 64% subclone EcoRI/HindIII fragment AE (0.419-0.525 map unit) grew into established but nontumorigenic lines. Transfection of EcoRI/HindIII AE-immortalized cells with a 4.4-kilobase Sac I/BamHI subfragment within BamHI E (0.554-0.584 map unit; overlaps the right-hand 16% of Bgl II C) converted them to tumorigenicity. The 4.4-kilobase subfragment encodes a 144-kDa protein immunologically and structurally similar to an infected cell protein designated ICP 10. DNA extracted from cells transformed with the 4.4-kilobase subfragment exhibited discrete hybridizing bands homologous to BamHI E fragment. Monoclonal antibody to ICP 10 precipitated a 144-kDa protein from the transformed cells and stained them in immunofluorescence. A tumor derivative established with the transformed cells did not stain with this antibody, but approximately equal to 25% of the cells stained with a monoclonal antibody to c-myc protooncogene products.
Point mutation generates constitutive expression of an inducible eukaryotic gene.
Authors: Sumrada R A , Cooper T G
Published: May 2006

We describe the analysis of two cis-dominant mutations that result in constitutive expression of the inducible CAR1 gene from yeast. One mutation results from insertion of a Ty element just upstream from the point where CAR1-specific transcription begins. The other mutation is a C-to-G transversion at position -153. Isolation of this point mutation, outside of the transcribed region of CAR1, suggests that expression of this gene is regulated at transcription. It also demonstrates the feasibility and usefulness of analyzing the regulatory sequences of eukaryotic genes on a nucleotide-by-nucleotide basis.