bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of anti-idiotype immunosorbents to isolate circulating antigen-specific T cell-derived molecules from hyperimmune sera.
Authors: Iverson G M , Eardley D D , Janeway C A , Gershon R K
Published: May 2006

We immunized four different sheep with antigen-binding material found in the serum of BALB/c mice 4 days after primary immunization with sheep erythrocytes (SRBC). The resultant antibodies made by the sheep contained a specificity(ies) that appeared to react with a dominant idiotype present on SRBC-specific Lyt-2+ T cells. The antiserum made by the sheep markedly inhibited the formation of antigen-specific rosettes by SRBC educated T cells but did not inhibit T cells educated to other heterologous erythrocytes from forming crossreacting rosettes with SRBC or specific rosettes with the homologous erythrocytes. The "anti-Id serum" was depleted of all activity against known immunoglobulin isotypes and light chains and then was used to isolate antigen-binding molecules from mice that were hyperimmunized with SRBC. The ShId+ material so isolated could be divided into two main groups--one that expressed immunoglobulin determinants, and one that did not. The former represented 15-25% of the ShId+ protein isolated and comprised a minority of the anti-SRBC antibody in the anti-SRBC serum; the latter group of proteins bound sheep glycophorin specifically and expressed constant region determinants found on a number of other antigen-specific T cell factors. These experiments suggest that antigen-binding molecules made by T cells display much less heterogeneity than do antibodies and also show that the serum of hyperimmune mice contains significant amounts of T cell-derived antigen-specific immunoregulatory molecules.
Inhibition of mammary tumor growth in rats and mice by administration of agonistic and antagonistic analogs of luteinizing hormone-releasing hormone.
Authors: Redding T W , Schally A V
Published: May 2006

Experiments were undertaken with estrogen-dependent mammary carcinomas in rats and mice to determine the antitumor activities of agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). Chronic administration of the agonist [D-Trp6]LH-RH or of antagonist 1 ( [NAc-D-p-Cl-Phe1,2-Phe3,D-Arg6-D-Ala10]LH-RH) at doses of 25 and 50 micrograms/day, respectively, for 21 days to mice bearing the MXT mammary carcinoma significantly decreased tumor weight and volume. The weight of the ovaries and serum progesterone levels in mice treated with [D-Trp6]LH-RH or antagonist 1 were also significantly reduced. In rats bearing the MT/W9A mammary adenocarcinoma, chronic administration of [D-Trp6]LH-RH at a dose of 25 micrograms twice a day or of antagonist 2 ( [NAc-D-p-Cl-Phe1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH) at a dose of 50 micrograms twice a day for 28 days significantly decreased tumor weight and volume. Chronic treatment with either [D-Trp6]LH-RH or antagonist 2 markedly diminished the weight of the ovaries and serum levels of both estrogen and progesterone. Serum luteinizing hormone was significantly decreased in rats treated with antagonist 2 but not in rats treated with [D-Trp6]LH-RH. There was a significant drop in serum prolactin levels in rats treated with [D-Trp6]LH-RH but not in those receiving antagonist 2. Regression of mammary tumors in rats and mice in response to chronic administration of [D-Trp6]LH-RH and the two antagonistic analogs of LH-RH suggests that these compounds should be considered for the development of a new hormone therapy for breast cancer in women.
Heavy metals induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle.
Authors: Abramson J J , Trimm J L , Weden L , Salama G
Published: May 2006

Micromolar concentrations of mercury, silver, and other reagents known to react with sulfhydryl groups are shown to stimulate ATPase activity and inhibit active calcium uptake in sarcoplasmic reticulum vesicles derived from rabbit fast skeletal muscle. These effects are caused by a dramatic increase in the calcium permeability of the sarcoplasmic reticulum. Measurements of Ca2+ permeability were made using both isotopes and by spectrophotometric techniques using the Ca2+ indicator arsenazo III. Air oxidation of a sulfhydryl group to a disulfide group also leads to a large increase in the calcium permeability of the sarcoplasmic reticulum.
Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.
Authors: Das H K , Biro P A , Cohen S N , Erlich H A , von Gabain A , Lawrance S K , Lemaux P G , McDevitt H O , Peterlin B M , Schulz M F , Sood A K , Weissman S M
Published: May 2006

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.
Two catabolite activator protein molecules bind to the galactose promoter region of
Escherichia coli
in the presence of RNA polymerase
Authors: Shanblatt Stephanie H. , Revzin Arnold
Published: May 2006

The catabolite activator protein (CAP) of
Escherichia coli
, complexed with cAMP, is required for efficient initiation of transcription from the galactose P1 promoter (start site at +1) but not from the overlapping P2 promoter (start site at -5) [Musso, R. E., DiLauro, R., Adhya, S. & deCrombrugghe, B. (1977)
Cell
12, 847-854]. We investigated the interactions between CAP/cAMP and the
gal
promoter region in the presence of RNA polymerase. DNase I protection experiments of
gal
promoter restriction fragments revealed that CAP/cAMP protects the DNA from digestion between positions -50 and -25 and that RNA polymerase protects it from -35 to +10; however,
gal
DNA in the presence of both CAP/cAMP and RNA polymerase is protected from DNase I digestion between positions -68 and +15. Results of exonuclease III protection experiments show that RNA polymerase alone protects the
gal
DNA from -30 to +15; when both CAP/cAMP and RNA polymerase are present in the reaction, protection is afforded from -65 to +20. We directly quantified the amount of cAMP and CAP bound to
gal
promoter DNA in the presence of RNA polymerase by selectively pelleting the ternary complexes (CAP/cAMP-RNA polymerase-
gal
promoter DNA) in a Beckman Airfuge. We found two CAP molecules specifically bound to the
gal
promoter, although only one cAMP molecule was found in the complex at low cAMP concentrations (but sufficient to support P1 transcription). Thus, both the DNA protection experiments and the centrifugation results indicate that RNA polymerase induces the binding of a second CAP molecule to the
gal
promoter in forming stable initiation complexes. It appears that the second CAP molecule is needed to stimulate initiation from the P1 promoter; this may be involved in regulating the relative rates at which transcription begins from the two
gal
start sites.
Clones of cytotoxic T lymphocytes reactive to haptenated allogeneic cells: precursor frequency and characteristics as determined by a split-culture approach.
Authors: Good M F , Nossal G J
Published: May 2006

CBA (H-2k) responder spleen cells have been cultured at limit dilution with trinitrophenyl (TNP)-modified BALB/c (H-2d) stimulator cells and a source of T-cell growth factor in order to generate cytotoxic effector clones. After culture, such clones were split into two to four replicates and each was assayed against a different target. This allowed identification of clones capable of lysing TNP-modified P815 (H-2d) targets but not unmodified P815 targets. Thus, clones specific for TNP and allogeneic restriction elements were detected without the need to use techniques that deplete the responder population of alloreactive cells. Cytotoxic T lymphocyte precursors (CTL-P) specific for TNP-modified P815 (major histocompatibility complex-nonidentical) targets were identified, at a low frequency (28.2 x 10(-6)) compared to CTL-P for TNP-modified C1.18 (H-2k) (identical) targets (224 x 10(-6)). The hapten specificity, H-2 restriction specificity, and Thy-1 status of these clones have been examined. Fourteen percent of CBA CTL-P reactive to TNP-modified P815 targets also showed reactivity to NIP-modified P815 targets, and 86% of CBA clones reactive to TNP-modified P815 targets ("allo-TNP-reactive" clones) failed to show reactivity to TNP-modified C1.18 targets--i.e., showed a restriction preference for allo rather than self. All such H-2d-restricted, TNP-specific clones were uniformly sensitive to anti-Thy-1 antibody and complement. Among the H-2k responders studied, we have not demonstrated CTL-P reactive to TNP-modified syngeneic cells which also react with H-2d cells or NIP-modified H-2d cells among 168 clones analyzed. This suggests that such clones, if present, are relatively rare.
Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus.
Authors: O'Connell A P , Urban M K , London W T
Published: May 2006

We tested the hypothesis that duck hepatitis B virus (DHBV) is a naturally occurring congenital infection of Pekin duck embryos. Of 219 embryos, 5-25 days after being laid, sera from 30 were found to be positive for endogenous DNA polymerase activity characteristic of hepatitis B-related viruses. The presence of the duck virus was confirmed by hybridization with cloned DHBV DNA. Viral DNA was also found in the livers of embryos incubated for 12 or 18 days. Electrophoretically different forms of DHBV DNA were identified in liver extracts that were not present in serum. These additional liver forms probably represent viral replication intermediates. These observations suggest that the vertical route is a major pathway of DHBV transmission and that viral replication may be initiated by the 12th day of embryonic life.
Immunocytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines.
Authors: Caceres A , Payne M R , Binder L I , Steward O
Published: May 2006

To determine whether dendritic spines contain actin, we evaluated the immunocytochemical localization of actin in the hippocampal formation and cerebral cortex of the rat. Monoclonal hybridoma antibodies were prepared against adult quail breast muscle actin. The culture supernatant of two cell lines (QAB1 and QAB2) was examined. Both antibodies bound only actin in crude brain homogenates, and neither exhibited species specificity. Electron microscopic analyses of sections reacted with QAB1 revealed staining of postsynaptic densities and dendritic microtubules but little staining of the cytoplasmic compartment of spines. However, sections reacted with QAB2 exhibited staining at the cytoplasmic compartment of spines as well as the sites stained by QAB1. We also evaluated the immunocytochemical distribution of beta-tubulin and high molecular weight microtubule-associated protein (MAP2) utilizing monoclonal antibodies. MAP2 was found in the dendritic spine as well as in the parent dendrite. However, beta-tubulin was found only in the postsynaptic density and in the microtubules of the parent dendrite. The combined results indicate that actin is present in the spine along with MAP2 and that there is a difference in the actin (or the state of actin) in the spine in comparison with other neuronal compartments.
Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.
Authors: Strader C D , Pickel V M , Joh T H , Strohsacker M W , Shorr R G , Lefkowitz R J , Caron M G
Published: May 2006

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.
5S rRNA gene transcription factor IIIA alters the helical configuration of DNA.
Authors: Reynolds W F , Gottesfeld J M
Published: May 2006

Relaxation of Xenopus 5S plasmid DNA (pX1o8) in the presence of transcription factor (TF) IIIA reduces the linking number of the DNA. Parallel experiments with plasmid pMB9 or cloned hepatitis B viral DNA indicate a degree of non-specific unwinding by TF; however, 60% of the effect observed for pX1o8 is due to specific interaction of TF IIIA with the 5S rRNA gene internal promoter sequence. The extent of unwinding (0.2-0.4 helical turn per TF IIIA binding site) is not consistent with the complete denaturation of the 50-base-pair TF binding site; however, it is consistent with a change in helix rotation, denaturation of 2-4 nucleotides per binding site, or DNA wrapping about a protein core. We show that proteins other than TF IIIA (bovine serum albumin and RNase) have no effect on the linking number of DNA when present during relaxation and that the unwinding activity associated with TF is heat labile. These results suggest that TF IIIA may facilitate transcription by altering the helical configuration of 5S DNA.
Cholesterol side-chain cleavage in the rat adrenal cortex: isolation of a cycloheximide-sensitive activator peptide.
Authors: Pedersen R C , Brownie A C
Published: May 2006

A cytosolic peptide activator (Mr approximately equal to 2,200) of cholesterol side-chain cleavage in the adrenal cortex has been isolated from normal corticotropin-treated rats and from rats implanted with the MtT/F4 corticotropin-secreting pituitary tumor. The isolation techniques were those common to peptide hormone purification, including tissue extraction into a highly acidic medium, gel filtration, and reverse-phase HPLC. The amino acid composition has been determined on acid hydrolysates. The activity of this adrenal peptide is acutely increased in hypophysectomized animals treated with corticotropin, and this increase is blocked by cycloheximide. The addition of activator peptide to adrenal mitochondrial preparations results in a rapid stimulation of pregnenolone formation that is dependent on activator concentration and a source of NADPH. In the absence of NADPH, addition of activator peptide to adrenal mitochondria increases the rate of cholesterol association with side-chain cleavage cytochrome P-450. The peptide therefore exhibits properties that are believed to characterize the hypothetical corticotropin-dependent labile activator of adrenal steroidogenesis.
Analysis of a break in chromosome 14 mapping to the region of the immunoglobulin heavy chain locus.
Authors: Linsley P S , Bech-Hansen N T , Siminovitch L , Cox D W
Published: May 2006

We have detected restriction fragment length polymorphisms associated with the immunoglobulin heavy chain C gamma genes. DNA from both parents of an individual having an unbalanced rearrangement of the long arm of chromosome 14, region q32 [Cox, D. W., Markovic, V. D. & Teshima, I. E. (1982) Nature (London) 297, 428-430], revealed distinctive patterns of BamHI fragments which hybridized with cloned probes from the C gamma 2-C gamma 4 gene cluster. The number of hybridizing fragments in both cases (five) equaled the number of known C gamma genes. Pedigree and densitometric analyses indicated that the proband did not have any maternal complement of C gamma gene-hybridizing fragments. Included on the deleted chromosomal segment was a C gamma gene having properties of the previously reported C gamma pseudogene. We also examined DNA from this family with a probe for the highly polymorphic locus D14S1, which recently was demonstrated to be tightly linked to the C gamma 1 gene locus [Balazs, I., Purrello, M., Rubinstein, P., Alhadeff, B. & Siniscalco, M. (1982) Proc. Natl. Acad. Sci. USA 79, 7395-7399]. EcoRI and EcoRI-BamHI fragments from both parents hybridized with a probe for this locus in DNA from the proband, indicating that, unlike the C gamma gene family, D14S1 was not deleted from the abnormal chromosome. Thus, the chromosomal breakpoint in the proband lies within region 14q32 between the two tightly linked markers, D14S1 and the C gamma 1 heavy chain gene locus. The D14S1 locus must lie proximal to the centromere relative to the C gamma gene family. The genetic variability detected with C gamma gene probes may prove useful for genetic analysis of structural rearrangements involving this region of chromosome 14.
H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.
Authors: Garner W H , Garner M H , Spector A
Published: May 2006

A 1-hr exposure of bovine lenses in organ culture to H2O2 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H2O2, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP concentrations decrease approximately equal to 10%, suggesting that glycolysis may be stimulated but ATP production is not able to keep up with the demand for energy. Examination of epithelial cell Mg2+-stimulated Na+,K+-ATPase isolated from the cultured lenses indicates H2O2-induced modification. At 5 mM MgATP, ATP hydrolysis is accelerated 30%; at 3 mM MgATP, hydrolysis is normal; and at 0.75 mM MgATP, it is inhibited 75%. p-Nitrophenyl phosphate hydrolysis and eosin maleimide binding indicate that K+ control of the enzyme is modified. Thus, a very early effect of H2O2 upon the lens, well before the formation of opacity, appears to be the uncoupling of Na+ and K+ transport from ATP hydrolysis.
High-frequency spontaneous mutation in the bacterio-opsin gene in Halobacterium halobium is mediated by transposable elements.
Authors: DasSarma S , RajBhandary U L , Khorana H G
Published: May 2006

We have recently characterized a transposable element, ISH1, which inactivates the bacterio-opsin (BO) gene in two purple membrane-deficient (Pum-) mutants of Halobacterium halobium. Examination of nine additional Pum- mutants now shows that in all of these the BO gene has been inactivated by insertion of one of two types of transposable elements. Four Pum- strains contain ISH1 within the BO gene, probably at the same site that we have previously characterized. A second element, ISH2, which is present in four more strains, inserts at multiple sites within the BO coding sequence. Significantly, another Pum- strain contains the ISH2 element 102 nucleotides upstream from the initiator codon for BO. ISH2, which is 520 nucleotides long, is the smallest insertion sequence known. Its sequence has been determined: it is A + T-rich (53%), contains a 19-base-pair inverted repeat at its termini, and, interestingly, duplicates either 10 or 20 base pairs at the target site during insertion. ISH2 is present in multiple copy numbers in the genome and contains several relatively short open reading frames.
Flow of mitochondrial DNA across a species boundary.
Authors: Ferris S D , Sage R D , Huang C M , Nielsen J T , Ritte U , Wilson A C
Published: May 2006

Restriction analysis shows that wild Scandinavian mice belonging to the species Mus musculus contain the mitochondrial DNA of a neighboring species, M. domesticus. This demonstration results from comparisons of Scandinavian mice with authentic M. domesticus and M. musculus from other parts of Europe. Electrophoretic and immunological analysis of eight diagnostic proteins confirms that mice from north of the hybrid zone in Denmark are M. musculus in regard to their nuclear genes. In contrast, the mice tested from this region and a nearby part of Sweden have exclusively M. domesticus types of mitochondrial DNA. Phylogenetic analysis of the restriction maps suggests that the mitochondrial DNAs found in Scandinavian M. musculus could stem from a single M. domesticus female.
High incidence of human type-C retrovirus (HTLV) in family members of a HTLV-positive Japanese T-cell leukemia patient.
Authors: Sarin P S , Aoki T , Shibata A , Ohnishi Y , Aoyagi Y , Miyakoshi H , Emura I , Kalyanaraman V S , Robert-Guroff M , Popovic M , Sarngadharan M , Nowell P C , Gallo R C
Published: May 2006

Sera and peripheral blood cells of an adult T-cell leukemia patient and several clinically normal members of his family from the northwest coast of Japan were examined for evidence of infection with human T-cell leukemia (lymphoma) virus (HTLV). The sera of the patient and his parents had antibodies to HTLV, whereas these antibodies were absent in the sera of the patient's brother and sister. T-cell lines were established from the peripheral blood lymphocytes of all of the family members, and all except the patient's sister expressed HTLV antigens (p19, p24, and reverse transcriptase) and type-C virus particles. Not only the fresh peripheral blood lymphocytes from the patient but also those from his clinically normal mother showed abnormal morphology of the kind characteristic of some patients with T-cell leukemia. These studies are consistent with previous evidence indicative of a high rate of HTLV infection within families, and they show that people whose sera are negative for antibodies may still be infected by HTLV. In addition, the results indicate that infection of T cells by HTLV can be associated with morphological transformation of the cells without other signs of leukemia.
Location and distribution of the light chains in clathrin trimers.
Authors: Kirchhausen T , Harrison S C , Parham P , Brodsky F M
Published: May 2006

Mouse monoclonal antibodies have been made that are specific for one of the light chains (LC-A) of calf brain clathrin. The determinant they recognize has been mapped by immunoelectron microscopy and shown to lie near the center of a clathrin trimer. Quantitative immunoprecipitation experiments with one of the monoclonal antibodies suggests that, despite the overall 1:2 stoichiometry, the light chain composition of an individual clathrin trimer is nearly random.
Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin.
Authors: Malik N , Miwa M , Sugimura T , Thraves P , Smulson M
Published: May 2006

Antibody to poly(ADP-ribose) has been covalently coupled to Sepharose and utilized to isolate selectively oligonucleosomes undergoing the poly(ADP-ribosyl)ation reaction from the bulk of chromatin. Approximately 12% of the unfractionated oligonucleosomes were bound to the immunoaffinity column and these represented essentially 100% of the original poly(ADP-ribosyl)ated nucleosomal species in the unfractionated chromatin. Poly(ADP-ribosyl)ated chromatin was not bound by preimmune IgG columns. KSCN eluted the modified nucleosomes in the form of nucleoprotein complexes. The eluted chromatin components were shown to contain poly(ADP-ribosyl)ated histones as well as automodified poly(ADP-ribose) polymerase. By using [3H]lysine- and [3H]arginine-labeled chromatin, it was shown that the poly-(ADP-ribosyl)ated histones, attached to stretches of oligonucleosomes bound to the column, had a 6-fold enrichment of the modification compared to histones of the unfractionated chromatin. This indicated that non-poly(ADP-ribosyl)ated nucleosomes, connected and proximal to the modified regions, were copurified by this procedure. This allowed characterization of the oligonucleosomal DNA around poly(ADP-ribosyl)ated chromatin domains to be compared with the unbound bulk chromatin. The data indicated that immunofractionated poly(ADP-ribosyl)ated oligonucleosomal DNA contained significant amounts of internal single-strand breaks compared with bulk chromatin. The bound nucleo-protein complexes were found to be enzymatically active for poly(ADP-ribose) polymerase after elution from the antibody column. In contrast, the unbound nucleosomes, representing 90% of the unfractionated chromatin, were totally inactive in the poly(ADP-ribosyl)ation reaction.
Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis.
Authors: Matts R L , Levin D H , London I M
Published: May 2006

The reticulocyte reversing factor (RF) isolated as a complex with eukaryotic initiation factor 2 (eIF-2) acts catalytically in restoring protein synthesis in reticulocyte lysates inhibited by heme deficiency. In reconstituted in vitro assay mixtures containing Mg2+ (0.25-0.5 mM), RF catalyzes the formation of the binary complex (eIF-2-GDP) but this effect is inhibited when eIF-2 is phosphorylated by the heme-regulated kinase for the alpha-subunit of eIF-2 (HRI). More significantly, RF catalyzes the rapid dissociation of (eIF-2-GDP), which permits the exchange of GTP for GDP and, in the presence of Met-tRNAf, promotes the formation of the ternary complex (eIF-2-Met-tRNAf X GTP). However, phosphorylation of the binary complex by HRI prevents its dissociation by RF and, as a consequence, ternary complex formation is inhibited. Our results indicate that phosphorylated binary complex [eIF-2(alpha P).GDP] interacts with RF to form a [RF . eIF-2(alpha P)] that is not readily dissociable. This binding of RF renders it unavailable to catalyze the dissociation of unphosphorylated binary complex, thereby blocking the recycling of eIF-2. Since RF is present in lysates at a limited concentration relative to that of eIF-2, the sequestering of RF in this manner could account for the observation that the phosphorylation of a small proportion of eIF-2 in heme-deficient lysates is sufficient to inhibit protein synthesis.
Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue.
Authors: Lipsky N G , Pagano R E
Published: May 2006

A fluorescent analogue of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-epsilon-aminocaproyl sphingosine (C6-NBD-ceramide), was used to investigate sphingolipid metabolism in Chinese hamster fibroblasts. C6-NBD-ceramide was incorporated into small unilamellar dioleoyl phosphatidylcholine vesicles and incubated with cells in monolayer culture at 2 degrees C, resulting in rapid and preferential transfer of the labeled ceramide from vesicles to cells. The cells were then washed and subsequently incubated at 37 degrees C for various intervals. The metabolism of C6-NBD-ceramide was monitored by lipid extraction and analysis, and the intracellular distribution of the labeled molecule was followed by fluorescence microscopy. Initially, fluorescence was detected almost exclusively in mitochondria, with over 90% of the extractable lipid fluorescence due to C6-NBD-ceramide. After 30 min at 37 degrees C, intense fluorescence appeared in the Golgi apparatus. This organelle was identified by colocalization of NBD fluorescence with a Golgi-apparatus-specific stain. At later times the plasma membrane became visibly labeled as well, at which point 90% of the cell-associated fluorescence was recovered as NBD-labeled sphingomyelin and NBD-labeled cerebroside. These metabolites were identified by enzymatic and biochemical analysis and by thin-layer chromatography of the fluorescent lipid extracts. The finding that C6-NBD-ceramide is used by these cells in standard pathways of sphingolipid biosynthesis suggests that this fluorescent precursor will be a valuable tool for correlating the metabolism of sphingolipids with their intracellular distribution and translocation. In addition, during its metabolism by Chinese hamster fibroblasts, this compound acts as a vital stain for the Golgi apparatus.
Coexpression of keratin- and vimentin-type intermediate filaments in human metastatic carcinoma cells.
Authors: Ramaekers F C , Haag D , Kant A , Moesker O , Jap P H , Vooijs G P
Published: May 2006

Metastatic tumor cells of epithelial origin present in effusions from human serous cavity fluids (ascites or pleural fluid) were examined for their intermediate-sized filament types by using antibodies to keratin, vimentin, and desmin in the indirect immunofluorescence technique. Solid epithelial tumors (both primary carcinomas and their metastases) contain keratin intermediate-sized filaments exclusively. However, when these cells are present in ascitic or pleural fluid, they also express vimentin, which occurs in a fibrillar organization. The possible effects of this additional, but temporary, cytoskeleton on metastatic growth or aggressiveness (or both) are discussed.
Identification of AAS genes and their regulatory role in general control of amino acid biosynthesis in yeast.
Authors: Penn M D , Galgoci B , Greer H
Published: May 2006

In yeast, most amino acid biosynthetic pathways are coregulated: starvation for a single amino acid results in derepression of enzyme activities for many different biosynthetic pathways. This phenomenon is referred to as "general control of amino acid biosynthesis." In this paper we describe the isolation and characterization of 43 amino acid analog-sensitive (aas-) mutants that are perturbed in this general regulatory system. These 43 mutations define four unlinked complementation groups, AAS101, AAS102, AAS103, and AAS104, two of which identify previously unreported genes involved in general control. These aas mutants are unable to derepress a number of amino acid biosynthetic genes, resulting in increased sensitivity to amino acid analogs, reduced growth rates, and reduced enzyme activity levels under amino acid starvation conditions. Thus, the AAS+ gene products function as positive regulatory elements for this system. We show that the AAS genes mediate these effects by regulating the mRNA levels of genes under their control.
Protection from spontaneous lymphoma development in SJL/J(v+) mice neonatally injected with dualtropic SJL-151 virus.
Authors: De Rossi A , D'Andrea E , Biasi G , Collavo D , Chieco-Bianchi L
Published: May 2006

In previous studies dualtropic type C retroviruses were isolated from spontaneous B-cell lymphomas that appear with a high incidence in SJL/J(v+) mice. In this report the possible in vivo pathogenic effect of one cloned dualtropic isolate, designated SJL-151, was investigated. SJL/J(v+) and CBA/J mice, neonatally injected with SJL-151 alone or in combination with SJL-ecotropic virus, were initially studied for virus recovery 4-8 weeks after infection by spleen cell cocultivation with mouse SC-1 and mink ML indicator cells. Whereas ecotropic virus was easily detected in treated mice, dualtropic virus was recovered only from the spleen cells of animals coinfected with SJL-ecotropic and SJL-151 viruses. With a panel of monoclonal antibodies the recovered dualtropic viruses showed an antigenic profile similar to that of the originally injected SJL-151 virus. Whereas virus-injected mice did not show lymphoma induction or acceleration, a remarkable decrease in spontaneous lymphoma incidence was observed in the SJL/J(v+) mice receiving SJL-151 virus alone. A virus-specific antibody response was detectable in these mice, but a similar serum reactivity was also demonstrated in SJL/J(v+) mice coinfected with SJL-ecotropic virus and SJL-151 virus, which subsequently developed lymphomas with the usual high incidence, thus rendering an antibody-mediated protective mechanism untenable. The possibility of viral interference, as an alternative mechanism for lymphoma prevention, is discussed in view of the findings that persistence of SJL-151 virus or de novo generation of dualtropic virus did not occur in aged SJL/J(v+) mice injected neonatally with SJL-151 virus alone.
Regulation of receptor capping in mouse lymphoma T cells by Ca2+-activated myosin light chain kinase.
Authors: Kerrick W G , Bourguignon L Y
Published: May 2006

Several characteristics of receptor capping in lymphocyte membranes suggest similarities with mechanisms underlying control of contraction in smooth muscle fibers. Both capping and contraction are Ca2+ dependent and require metabolic energy. Contractile proteins such as actin and myosin are associated with the cap, as is calmodulin, which mediates the Ca2+ dependence of smooth muscle contraction. Recent studies have shown that myosin light chain kinase (MLCK), which plays a central role in regulation of smooth muscle contraction, is also present in isolated lymphocyte membrane-cytoskeleton complexes. We have explored this analogy further, using mouse lymphoma T cells whose membranes were rendered permeable to small proteins by using a low-Ca2+ EGTA solution similar to that used to chemically skin smooth muscle cells. Permeabilized lymphocytes were then exposed to solutions containing various combinations of high or low Ca2+, ATP, or other nucleotides (5'-adenylyl imidodiphosphate, adenosine 5'-[gamma-thio]triphosphate, guanosine 5'-[gamma-thio]triphosphate, CTP, ITP, UTP, and GTP), calmodulin, Ca2+-insensitive MLCK (MLCK subunit that has been stripped of the Ca2+ binding site), and the catalytic subunit of cAMP-dependent protein kinase that phosphorylates (and thereby inactivates) MLCK. Capping of concanavalin A-labeled receptors in these various test solutions was scored. In all solutions the capping observed in permeable lymphoma cells correlated well with contraction previously observed in similarly treated skinned smooth muscle fibers, providing strong evidence for the involvement of myosin light chain phosphorylation in the regulation of receptor capping.
Protective immunization of the squirrel monkey against asexual blood stages of Plasmodium falciparum by use of parasite protein fractions.
Authors: Dubois P , Dedet J P , Fandeur T , Roussilhon C , Jendoubi M , Pauillac S , Mercereau-Puijalon O , Pereira Da Silva L
Published: May 2006

We had previously shown that two polypeptides of Plasmodium falciparum are preferentially recognized by antibodies of resistant squirrel monkeys Saimiri sciureus. Free parasites were isolated from synchronized cultures on human erythrocytes initially inoculated with infected Saimiri erythrocytes. Crude extracts were prepared from mature schizont stages and electrophoresed on preparative NaDodSO4/polyacrylamide gels. Two groups of five monkeys were immunized with protein fractions eluted from the 75- and 100-kilodalton regions of the gels. Strong protection against challenge by the homologous strain of P. falciparum was induced in both groups. Analysis of specific anti-malarial antibodies revealed a homogeneous response of all the animals against a few polypeptides of the mature parasite.
Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype induced by the adenosine deaminase inhibitor 2'-deoxycoformycin.
Authors: Hershfield M S , Kurtzberg J , Harden E , Moore J O , Whang-Peng J , Haynes B F
Published: May 2006

Selective failure of lymphoid development occurs in genetic deficiency of adenosine deaminase (ADA). We examined the in vivo effects of a potent inhibitor of ADA, 2'-deoxycoformycin, which was used to treat a patient with refractory acute leukemia. Unexpectedly, within 7 days of starting treatment, the leukemic phenotype underwent complete conversion from T lymphoblastic to promyelocytic, with kinetics that suggested a precursor-product relationship between the two cell populations. Pretreatment T lymphoblasts and posttreatment promyelocytes had the same abnormal karyotype. Upon culture in vitro, the former transformed spontaneously over several weeks into mature myeloid cells. We conclude that the leukemia arose from a multipotent stem cell capable of both lymphoid and myeloid differentiation. Effects of ADA inhibition on leukemia cells during treatment included expansion of the deoxyadenosine nucleotide pool and accumulation of S-adenosylhomocysteine, a potent inhibitor of S-adenosylmethionine-dependent methylation. The influence of these changes on the leukemic phenotype is discussed in terms of (i) selective cytotoxicity to T lymphoblasts, which accumulated deoxyadenosine nucleotides more efficiently than did the patient's promyelocytes during in vitro incubation with deoxycoformycin plus deoxyadenosine, and (ii) induction of an altered program of differentiation.
Structural analysis of fibronectin and its collagen-binding fragment from several cell lines.
Authors: Ali I U
Published: May 2006

Tryptic peptide mapping of fibronectin from various species indicates that (i) there exists a remarkable similarity between methionine-labeled tryptic peptides of fibronectins isolated from cells of widely separated species such as human, chicken, mouse, hamster, and rat; (ii) fibronectins from normal and transformed or tumor cells of the same species are very similar, if not identical; and (iii) collagen-binding regions of fibronectin from different species share identical major peptides.
Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA.
Authors: Law S W , Brewer H B
Published: May 2006

The cDNA clones encoding the precursor form of human liver apolipoprotein A-I (apoA-I), preproapoA-I, have been isolated from a cDNA library. A 17-base synthetic oligonucleotide based on residues 108-113 of apoA-I and a 26-base primer-extended, dideoxynucleotide-terminated cDNA were used as hybridization probes to select for recombinant plasmids bearing the apoA-I sequence. The complete nucleic acid sequence of human liver preproapoA-I has been determined by analysis of the cloned cDNA. The sequence is composed of 801 nucleotides encoding 267 amino acid residues. PreproapoA-I contains an 18-amino-acid prepeptide and a 6-amino-acid propeptide connected to the amino terminus of the 243-amino acid mature apoA-I. Southern blotting analysis of chromosomal DNA obtained from peripheral blood indicated the apoA-I gene is contained in a 2.1-kilobase-pair Pst I fragment and there is no gross difference in structural organization between the normal apoA-I gene and the Tangier disease apoA-I gene.
Specific factor conferring nuclease hypersensitivity at the 5' end of the chicken adult beta-globin gene.
Authors: Emerson B M , Felsenfeld G
Published: May 2006

Chromatin in the 5' flanking region of the transcriptionally active chicken adult beta(beta A) globin gene is hypersensitive to nuclease digestion. When plasmids carrying this gene are combined with histones in the presence of extracts from 9-day-old chicken erythrocyte nuclei, the resultant complex displays hypersensitive behavior in the same region. No such behavior is observed with extracts from cells in which the beta A-globin gene is not expressed. Partially purified preparations of the factor conferring hypersensitivity bind preferentially to DNA fragments containing the hypersensitive region.
Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12.
Authors: Ferrara P , Duchange N , Zakin M M , Cohen G N
Published: May 2006

In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine. An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology. These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.
Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A.
Authors: Winkler M A , Merat D L , Tallant E A , Hawkins S , Cheung W Y
Published: May 2006

The calmodulin-dependent protein phosphatase from bovine brain is composed of two subunits: subunit A, Mr 60,000, and subunit B, Mr 16,500. Using in vitro immunization techniques, we have produced a monoclonal antibody specific for the phosphatase. The antibody was immobilized to Sepharose 4B to prepare an immunoabsorbent column, which was used to purify the enzyme. Phosphatase isolated from the column showed a polypeptide with Mr 60,000, equivalent to subunit A, which showed calmodulin-dependent phosphatase activity. Subunit B was not obtained from the column. Limited trypsin digestion stimulated phosphatase activity, yielding polypeptides of Mr 59,000, 43,000, and 16,000; the phosphatase activity after trypsin digestion was calmodulin independent. Chromatography of trypsin-treated phosphatase on an immunoaffinity column yielded two proteins, Mr 59,000 and 43,000, that were catalytically active and calmodulin independent. In a separate experiment, the two subunits of the phosphatase were separated by gel filtration in 6 M urea. Subunit A isolated from the filtration column showed little or no activity in the presence of Ca2+ and calmodulin, but it showed calmodulin-dependent phosphatase activity in the presence of 0.8 mM Mn2+. Subunit B was catalytically inactive. Collectively, these results indicate that subunit A and its proteolytic fragment contain the catalytic site and the antigenic determinant for the monoclonal antibody.
Expandable var1 gene of yeast mitochondrial DNA: in-frame insertions can explain the strain-specific protein size polymorphisms.
Authors: Hudspeth M E , Vincent R D , Perlman P S , Shumard D S , Treisman L O , Grossman L I
Published: May 2006

The var1 locus of yeast mitochondrial DNA encodes a protein of the small mitochondrial ribosome subunit, denoted var1 protein. The size of var1 protein was previously shown to exhibit a strain-dependent polymorphism, determined by various combinations of at least three genetic elements. We report here that the var1 gene is itself polymorphic and that the six forms of this gene examined here differ by various combinations of three in-frame insertions into the coding region of the smallest allele. These insertions, which appear to be the molecular basis for the genetic elements, could increase the size of var1 protein by 8, 10, 16, 24, or 26 amino acid residues, accounting for the observed protein polymorphisms. Furthermore, we have characterized three additional sources of sequence variation located outside of the coding region but within major transcripts of the var1 gene.
Translocated c-myc oncogene of Burkitt lymphoma is transcribed in plasma cells and repressed in lymphoblastoid cells.
Authors: Croce C M , Erikson J , ar-Rushdi A , Aden D , Nishikura K
Published: May 2006

We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid cells or mouse plasmacytoma cells for the expression of the translocated c-myc oncogene. The results of this study indicate that the translocated c-myc oncogene is transcribed in plasma cells but is repressed in lymphoblastoid cells. Thus, the factors necessary for translocated c-myc transcription are present in plasma cells and Burkitt lymphoma cells but are absent or inactive in lymphoblastoid cells. Since the distance between the rearranged immunoglobulin loci and the c-myc oncogene can even exceed 30-50 kilobases, we speculate that the translocated c-myc oncogene is under the transcriptional control of enhancer-like elements capable of acting over long distances. The activity of this long-range enhancer may depend on the interaction with transacting factors that are active in plasma cells and in Burkitt lymphoma cells but are not active in lymphoblastoid cells. We also examined the transcription of the first exon of the c-myc oncogene, which becomes separated from the second and third exon because of the chromosomal break involving the first intron. This exon is transcribed at high levels in ST486 Burkitt lymphoma cells with the t(8;14) chromosome translocation. Hybrids between lymphoblastoid and ST486 cells expressed high levels of transcripts of the first exon, whereas hybrids between plasma cells and ST486 cells did not. Thus, transcription of the separated first exon can be enhanced in lymphoblastoid and Burkitt lymphoma cells because of its close proximity to the heavy chain enhancer that is normally located between the joining and the switch region of the C mu gene. Such enhancement, however, does not occur in plasma cells, possibly because these cells are able to suppress completely the c-myc oncogene, unless it has been placed in the proximity of a rearranged immunoglobulin constant region gene.
Synaptic stimulation alters protein phosphorylation
in vivo
in a single
Aplysia
neuron
Authors: Lemos José R. , Novak-Hofer Ilse , Levitan Irwin B.
Published: May 2006

Protein phosphorylation was examined in the identified
Aplysia
neuron R15,
in vivo
, after the intracellular injection of [γ-
32
P]ATP. Two-dimensional gel electrophoretic analysis indicates that at least 70 proteins are phosphorylated within R15 during a 50-min labeling period. Application of serotonin (5HT) results in an increase in K
+
conductance in R15 and a concomitant change in the phosphorylation pattern: there are increases or decreases in the phosphorylation of some proteins, and at least five phosphoproteins appear that are not observed in control cells. Dopamine causes a decrease in voltage-dependent inward conductance in R15 and also alters the phosphorylation pattern: several of the phosphorylation changes are similar to those produced by 5HT, while others are unique to dopamine. Stimulation of the branchial nerve leading to the abdominal ganglion results in a long-lasting synaptic hyperpolarization of R15. The conductance changes underlying this response include an increase in K
+
conductance (identical to that produced by 5HT) together with a decrease in voltage-dependent inward conductance (identical to that produced by dopamine). The phosphorylation changes induced in R15 by branchial nerve stimulation resemble a combination of the changes induced by 5HT and dopamine. The results demonstrate that synaptic stimulation can modulate the phosphorylation of specific proteins in a single identified postsynaptic neuron and are consistent with the hypothesis that protein phosphorylation can regulate the regulate the activity of neuronal ion channels.