bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming growth factor(s) production enables cells to grow in the absence of serum: an autocrine system.
Authors: Kaplan P L , Anderson M , Ozanne B
Published: May 2006

Kirsten murine sarcoma virus (KiMSV)-transformed rat-1, normal rat kidney (NRK), and BALB/c 3T3 cells are capable of continual growth in a serum-free medium supplemented with transferrin and insulin but with no exogenous mitogenic growth factors. Cells transformed by a mutant of KiMSV that is temperature sensitive for the maintenance of transformation grow in this medium at the permissive temperature only. At the nonpermissive temperature, growth is dependent upon the presence of serum-free conditioned medium from the transformed cells. Normal rat-1 cells are also dependent upon factors from the transformed cells for growth in this serum-free/mitogen-free medium. The serum-derived growth factors, epidermal growth factor, and fibroblast growth factor have no effect on the transformed cells, although epidermal growth factor can replace transforming growth factors produced by KiMSV-transformed cells for the growth of rat-1 cells. Growth of the transformed cells in serum-free medium at clonal densities is dependent upon the presence of conditioned medium collected from the same cells grown to high densities. These results show that (i) growth in serum-free/mitogen-free medium is a general property of KiMSV-transformed cells and (ii) growth of the transformed cells in this medium is dependent upon the presence of growth factors known to be produced by the cells, and they provide support for the hypothesis that serum-free growth of KiMSV-transformed cells is dependent upon ectopically produced growth factors.
Identification of Epstein-Barr virus strain differences with monoclonal antibody to a membrane glycoprotein.
Authors: Qualtiere L F , Chase R , Vroman B , Pearson G R
Published: May 2006

Two monoclonal antibodies directed against Epstein--Barr virus (EBV)-induced membrane antigens (MA) were isolated in this study. On of the monoclonal antibodies, designated 2F5.6, was an IgG2 which, as detected by membrane and fixed cell immunofluorescence, reacted with MA-positive lymphoblastoid cell lines that produced transforming EBV but not with the MA-positive P3HR-1 cell line that produced the lytic, nontransforming strain of this virus. This antibody precipitated the Mr 320,000/350,000 glycoprotein from B-95 virus infected cultures and the Mr 300,000 and 220,000/250,000 glycoproteins from Raji cells superinfected with P3HR-1 virus but did not precipitate any of these EBV-specific glycoproteins from the P3HR-1 cell line. In contrast, the second monoclonal antibody, IgM designated B10.3, reacted with all virus-producing cell lines including the P3HR-1 cell line. The identity of the glycoprotein that serves as the target for this antibody is still unknown. Neither antibody had neutralizing activity against the B-95 or P3HR-1 strain of EBV. These results indicated that the 2F5.6 monoclonal antibody was directed against an antigenic determinant on the major membrane glycoprotein which is common to transforming strains of EBV but absent from the lytic P3HR-1 stain whereas the B10.3 monoclonal antibody was directed against a group-specific EBV-induced membrane determinant.
Specific differentiation events induced by erythropoietin in cells infected in vitro with the anemia strain of Friend virus.
Authors: Koury M J , Bondurant M C , Duncan D T , Krantz S B , Hankins W D
Published: May 2006

Mouse bone marrow cells infected in vitro with the anemia strain of Friend leukemia virus from large clusters (bursts) of erythroblasts after 5 days in culture in methylcellulose medium. Two types of erythroblast populations can be isolated from bursts of infected cells by manipulation of the culture conditions. One type of erythroblast, which is obtained when erythropoietin (EP) is added to the culture, has proliferated and undergoes differentiation to become an erythrocyte. The second type of erythroblast, which is obtained when no EP is added to the culture, is the product of extensive proliferation, but it fails to undergo the terminal stages of erythroblast differentiation. Comparisons of these two types of erythroblasts demonstrate that specific EP effects include changes in the nucleus, cytoplasm, and membrane of the treated cells. Those events of erythroid differentiation shown to be directed by EP were extrusion of the nucleus from the erythroblast, induction of uroporphyrinogen I synthetase activity, increased iron incorporation into protoporphyrin, synthesis of alpha- and beta-globin polypeptides due largely to increased mRNA production, and synthesis and incorporation of spectrin into the cell membrane. In this system, EP promotes these effects without observable stimulation of progenitor proliferation in addition to that caused by the virus alone. Thus, the role of EP in terminal erythrocyte differentiation is not simply that of an erythroid-specific mitogen.
Gut-mediated hypercalcemia in rabbits bearing VX2 carcinoma: new mechanism for tumor-induced hypercalcemia.
Authors: Doppelt S H , Slovik D M , Neer R M , Nolan J , Zusman R M , Potts J T
Published: May 2006

The VX2 carcinoma-bearing rabbit is an animal model for tumor-induced hypercalcemia, thought to be due to increased bone destruction effected by prostaglandin E2. The present experiments suggest that the pathophysiology of the hypercalcemia differs from that previously proposed. Tumor was transplanted intramuscularly into 2.5- to 3-kg male New Zealand White rabbits, which were conditioned to a 1.5% calcium diet and treated with daily subcutaneous injections of dichloromethane diphosphonate (10 mg . kg-1 . day-1), a potent inhibitor of bone resorption, or 0.9% NaCl (2 ml . kg-1 . day -1). The diphosphonate had no significant effect on plasma Ca2+ in either group. After day 31, half the animals of each group were fed a calcium-free diet. This normalized the plasma Ca2+ in each VX2-bearing rabbit within 3 to 4 days but had little effect in control rabbits. In a second series of experiments, VX2-bearing rabbits maintained on standard rabbit chow were treated for 11 days with parenteral indomethacin (30--60 mg/day) or 0.9% NaCl. Although indomethacin normalized the markedly elevated urinary excretion of prostaglandin E2, both treatment groups became severely hypercalcemic. Dietary calcium restriction promptly restored to normal the plasma Ca2+ concentration. In a third series of experiments, rabbits were fed standard rabbit chow and treated with oral indomethacin (40 mg/day) while control-rabbits were pair fed in identical chow. Transplantation of VX2 tumor into both groups caused hypercalcemia. We conclude that the hypercalcemia produced by this tumor strain is indomethacin resistant and dependent on an increase in gastrointestinal calcium absorption, not on skeletal calcium mobilization.
Immunocytochemistry with internally labeled monoclonal antibodies.
Authors: Cuello A C , Priestley J V , Milstein C
Published: May 2006

One of the advantages of the production of monoclonal antibodies by tissue culture methods is that they can be internally labeled by using appropriate radioactive amino acid in the culture fluid. Thus, radioactive immunological probes of high specific activity can be prepared. Here we report applications of these internally labeled monoclonal antibodies for the direct localization of immunoreactive sites in the central nervous system of the rat at both light and electron microscopic levels (radioimmunocytochemistry). We explored the combined use of radioimmunocytochemistry and immunoenzymatic methods for the simultaneous detection of two antigenic sites: substance P and serotonin or substance P and enkephalin. In neurobiology this procedure could help to clarify certain aspects of transmitter-specific synaptic interactions and the coexistence of neuroactive substances in single neuronal cell bodies or nerve terminals. We also describe the application of radioimmunocytochemistry with internally labeled monoclonal antibodies to quantify the immunoreactions in discrete microscopic areas.
Physical separation and characterization of two types of benzodiazepine receptors.
Authors: Lo M M , Strittmatter S M , Snyder S H
Published: May 2006

Two distinct benzodiazepine receptors are solubilized differentially by various detergents. The receptor sites that resist solubilization, designated type I, are most highly concentrated in the cerebellum and corpus striatum whereas the more readily solubilized receptors, type II, are most enriched in the hippocampus. The type I receptors display higher affinity for beta-carboline esters and a triazolopyridazine whereas several benzodiazepines do not differentiate the two receptors. The type I receptors can be solubilized with 2% Triton X-100/1 M NaCl; they retain the same drug specificity as in the particulate state.
Binding properties of a cell adhesion molecule from neural tissue.
Authors: Rutishauser U , Hoffman S , Edelman G M
Published: May 2006

We have previously identified and purified a cell surface glycoprotein from retina and brain, called neural cell adhesion molecule or N-CAM, that appears to be involved in neural cell--cell adhesion, the fasciculation of neurites, and the formation of normal tissue patterns in the retina. The present studies reveal that artificial vesicles containing lipid and purified N-CAM bind to different cell types with a specificity similar to that of nerve cells. The same results were obtained with soluble N-CAM that had been briefly exposed to pH 3. In both cases the binding altered the rate of aggregation of neural cells and, like cell--cell adhesion, was inhibited by antibodies against N-CAM. The results support the proposal that N-CAM is a ligand in the formation of bonds between nerve cell membranes. Moreover, results of studies of vesicle--vesicle interactions and of N-CAM binding to cells coated with anti-(N-CAM) Fab' fragments were consistent with the idea that the N-CAM molecules on different cells may interact directly to form cell--cell bonds.
Bioassay for components regulating eukaryotic gene expression: a chromosomal factor involved in the generation of histone mRNA 3' termini.
Authors: Stunnenberg H G , Birnstiel M L
Published: May 2006

We have adapted the oocyte injection procedure for the detection of regulatory components involved in the transcription of a eukaryotic mRNA gene. Injection of the histone gene repeat h22 DNA of Psammechinus miliaris into the Xenopus oocyte nucleus results in correct initiation of the histone mRNAs, but readthrough by RNA polymerase occurs at the 3' end of the H3 histone gene (Hentschel, C. C., Probst, E. & Birnstiel, M. L. (1980) Nature (London) 288, 100-102). Coinjection into the oocyte of a chromosomal salt wash fraction derived from sea urchin embryos results in the generation of authentic 3' termini of the histone H3 mRNA. We have partially purified the protein component by column chromatography and density gradient centrifugation. The regulatory factor binds to heparin columns and, hence, has the properties anticipated of an RNA- or DNA-binding protein. The sedimentation coefficient of the active component was determined to be about 12 S, suggesting a molecular weight of 200,000-250,000.
Opal suppressor serine tRNAs from bovine liver form phosphoseryl-tRNA.
Authors: Hatfield D , Diamond A , Dudock B
Published: May 2006

An unusual minor species of bovine liver serine tRNA has previously been isolated, sequenced, and found to suppress the UGA termination codon in protein synthesis in vitro [Diamond, A., Dudock, B. & Hatfield, D. (1981) Cell 25, 497-506]. We have now found that this tRNA can be a substrate in a specific phosphorylation reaction in which phosphoseryl-tRNA is formed. Moreover, bovine liver contains a second UGA suppressor serine tRNA (tRNASerNCA; N is a modified nucleoside) which also forms phosphoseryl-tRNA. The nucleotide sequence and coding properties of tRNASerNCA are presented.
Structures of the genes for the β and ε subunits of spinach chloroplast ATPase indicate a dicistronic mRNA and an overlapping translation stop/start signal
Authors: Zurawski Gerard , Bottomley Warwick , Whitfeld Paul R.
Published: May 2006

A 2,4-kilobase-pair region of spinach chloroplast DNA adjacent to the gene for the large subunit of ribulosebisphosphate carboxylase has been analyzed by RNA hybridization,
in vitro
transcription/translation, and DNA sequence determination. The analysis indicates that this region carries the genes for the β and ε subunits of chloroplast ATPase and that the two genes are cotranscribed into a dicistronic mRNA with 4-base-pair overlap between the stop codon of the β-subunit gene and the start codon of the ε-subunit gene. The ATPase and carboxylase genes are transcribed divergently with respect to each other. The deduced amino acid sequences of the β and ε subunits from spinach show 67% and 26% homology, respectively, with the published sequences of the β and ε subunits of
Escherichia coli
ATPase.
Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.
Authors: Hatzfeld J A , Hatzfeld A , Maigne J
Published: May 2006

Purified fibrinogen at concentrations of 3-30 nM has been found to stimulate continuous growth of human lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphoma-derived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 10(5) cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, approximately equal to 50 granulocyte-macrophage colonies per 10(5) cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.
Excluded-volume effect on quasi-elastic light scattering by flexible macromolecules
Authors: Tanaka Genzo , Stockmayer Walter H.
Published: May 2006

First-order perturbation expressions are developed for the first cumulant (initial time derivative) of the dynamic structure factor observable by photon-correlation measurements of the light scattered by flexible chain molecules in solution. A dimensionless coefficient
C
, which measures the initial departure of the first cumulant from proportionality to the square of the scattering vector, is found to be only slightly altered by excluded-volume effects.
Spermidine-condensed phi X174 DNA cleavage by micrococcal nuclease: torus cleavage model and evidence for unidirectional circumferential DNA wrapping.
Authors: Marx K A , Reynolds T C
Published: May 2006

Spermidine-condensed phi X174 replicative form (RF) II DNA was digested with micrococcal nuclease to yield seven identifiable DNA bands forming an arithmetic fragment-length series. The DNA monomer unit length was found to be 780 +/- 80 base pairs. This result is most consistent with a proposed model for micrococcal nuclease cleavage of a DNA torus organized by the unidirectional, circumferential wrapping of B-geometry DNA. By a topological consideration, the blunt-end-rod-fusion model for torus formation [Eickbush, T. H. & Moudrianakis, E. N. (1978) Cell 13, 295-306] is shown to be inconsistent with our empirical solution results. We propose a continuous, circumferential DNA wrapping model in which a significant fraction of the collapsed circular phi X174 RFII DNA molecules form regular toruses comprised of seven complete, unidirectional double-helical wraps.
Evidence for multiple origins of the beta E-globin gene in Southeast Asia.
Authors: Antonarakis S E , Orkin S H , Kazazian H H , Goff S C , Boehm C D , Waber P G , Sexton J P , Ostrer H , Fairbanks V F , Chakravarti A
Published: May 2006

To investigate whether recurrent mutation has contributed to the high frequency of the beta E-globin gene in Southeast Asia, we used the haplotypes at three polymorphic restriction sites within and to the 3' side of the beta-globin gene to predict the framework of 23 beta E-globin genes. These haplotypes suggested that beta E-globin genes are present in two different beta-globin gene frameworks. DNA sequence determination of one gene representing each framework demonstrated that the same mutation (GAG leads to AAG at codon 26) was present in both frameworks. Moreover, the frameworks differed at three nucleotide positions known to be polymorphic in Mediterraneans. These polymorphic sites are located 70 nucleotides to the 5' side of the beta E mutation and 382 and 1032 nucleotides to the 3' side of it. The existence of the beta E mutation in these two beta-globin gene frameworks can be explained by (i) recurrent mutation giving rise to beta E-globin, (ii) a double crossing-over event, or (iii) two single crossing-over events. Mathematical analysis suggests that the first alternative, recurrent mutation of G leads to A at the first nucleotide of codon 26, is most likely.
T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood.
Authors: Albertini R J , Castle K L , Borcherding W R
Published: May 2006

Rare thioguanine-resistant T lymphocytes, present in vivo in human peripheral blood, were isolated and grown in vitro as thioguanine-resistant cultured T cells. The conditions for their selection in vitro were such that thioguanine resistance had to have arisen in vivo. The mutant cells bore T-cell surface markers, maintained their thioguanine resistance in vitro in the presence or absence of selection, and were deficient in hypoxanthine-guanine phosphoribosyltransferase activity.
Human type I procollagen genes are located on different chromosomes.
Authors: Huerre C , Junien C , Weil D , Chu M L , Morabito M , Van Cong N , Myers J C , Foubert C , Gross M S , Prockop D J , Boué A , Kaplan J C , de la Chapelle A , Ramirez F
Published: May 2006

A recombinant plasmid containing sequences complementary to human pro-alpha l(I) collagen mRNA was used for the chromosomal assignment of the pro-alpha l(I) collagen gene. Restriction endonuclease analysis of DNA from mouse-human and Chinese hamster-human somatic cell hybrids revealed cosegregation with human chromosome 17. Hybrids containing derivative chromosomes with a t(2;17)(q14;q21) translocation showed cosegregation of the pro-alpha l(I) gene with the segment 17q21 leads to qter. In situ hybridization on human metaphasic chromosomes confirmed this conclusion.
Purification, primary structure, and homology relationships of a chloroplast ribosomal protein
Authors: Bartsch Marius , Kimura Makoto , Subramanian Alap-Raman
Published: May 2006

A chloroplast ribosomal protein that showed immunological homology to
Escherichia coli
ribosomal protein L12 was purified from spinach (
Spinacia oleracea
) leaves and its primary structure was determined by manual micro Edman degradation. The protein is composed of 130 amino acid residues and has
M
r
13,576. It shows structural features characteristic of the L12 proteins of eubacterial 70S ribosomes (e.g., identical amino acid residues in about 50% of the sequence) but no apparent homology to the L12-type proteins of eukaryotic cytoplasmic 80S ribosomes. The homology to eubacterial proteins is highest in the COOH-terminal region (70%) and low in the NH
2
-terminal region (<20%).
Synapse formation in response to estrogen in the medial amygdala developing in the eye.
Authors: Nishizuka M , Arai Y
Published: May 2006

Medial amygdaloid tissue, taken from female rats immediately after birth, was transplanted into the anterior chamber of the eye in adult ovariectomized host rats in order to elucidate the influence of estrogen on synapse formation without contribution of neural afferents. After injections of estradiol benzoate or oil vehicle to the hosts for 20 successive days, the grafts were processed for semiquantitative electron microscopic study to examine synaptic density in the neuropil. The number of synapses on dendritic shafts vs. dendritic spines was not significantly different in the control group. In contrast, in the grafts exposed to estrogen, shaft synapses occurred more frequently than spine synapses. Synaptic density on shafts was significantly greater in these grafts than that in the controls, although the density on spines did not differ between the two groups. These data show that estrogen affects the medial amygdaloid neurons themselves and specifically facilitates the formation of dendritic shaft synapses in oculo. Our previous report raises the possibility that the specific increase of shaft synapses induced by sex steroids is involved in the process of sexual differentiation of neuronal networks from the inherently feminine pattern to the masculine pattern in the medial amygdala. Therefore, the present findings may provide evidence that sexual differentiation triggered by sex steroids is accomplished by intrinsic factors in the neurons of the medial amygdala.
Coexistence in human and primate neuromuscular junctions of enzymes synthesizing acetylcholine, catecholamine, taurine, and gamma-aminobutyric acid.
Authors: Chan-Palay V , Engel A G , Wu J Y , Palay S L
Published: May 2006

Coexistence of neurotransmitter-synthesizing enzymes choline acetyltransferase, cysteine sulfinic acid decarboxylase, tyrosine hydroxylase, and L-glutamic acid decarboxylase was demonstrated at human and primate neuromuscular junctions with specific antibodies directed against these enzymes. Motor end plates were identified in unfixed cryostat sections by standard cholinergic markers for acetylcholinesterase and the acetylcholine receptor. Each of the four transmitter-synthesizing enzymes was localized at end plates displaying these markers. The presence of any two of the four enzymes at a given end plate was established by (i) showing immunoreaction for one enzyme followed by elution and demonstration of immunoreaction for a second enzyme, and (ii) paired immunofluorescence with simultaneous demonstration of one enzyme with a rhodamine-labeled second antibody and of the other enzyme with a fluorescein-labeled second antibody. These findings imply that motor nerve terminals have the capacity for synthesizing not only acetylcholine but also taurine, catecholamines, and gamma-aminobutyric acid. These substances, in turn, may participate in the normal regulation of nerve-muscle interaction or be significant in specific disorders involving the motor unit.
Embryonic to adult conversion of neural cell adhesion molecules in normal and staggerer mice.
Authors: Edelman G M , Chuong C M
Published: May 2006

The neural cell adhesion molecule (N-CAM) has an unusually high amount of sialic acid (28-35 g/100 g of polypeptide) and shows microheterogeneity in electrophoretic gels in its embryonic or E form. During development, the molecule undergoes conversion to several adult or A forms, which resemble the E form but which on the average have only 10% sialic acid and do not appear to be microheterogeneous. In the present study, rabbit antibodies to mouse N-CAM and two different monoclonal antibodies were used to follow the E leads to A conversion in normal and mutant mice. E leads to A conversion to three forms (Mr 180,000, Mr 140,000, and Mr 120,000) was found to occur at different rates in different parts of the brains of wild-type mice. Examination of the entire cerebellum of the granuloprival mouse mutant staggerer (sg/sg) showed that the E leads to A conversion did not occur by 21 days after birth, whereas in wild type it was almost complete at that time. There was also some delay in E leads to A conversion within the cerebral cortex of sg/sg, although phenotypically no evidence of cortical disorder has been detected. In pooled tissues from phenotypically normal-appearing littermates, (i.e., a mixture of sg/+ and +/+), a slight conversion delay was also found in cerebellum and cortex. The mutants weaver, reeler, and jimpy all showed normal schedules of E leads to A conversion. These observations raise the possibility that a major defect in staggerer mutants relates to a failure in local surface modulation of N-CAM to produce the A forms of the molecule. Some of the failures of synapse formation and of cell survival seen in this disease may result from the anomaly, which is likely to alter the binding properties of N-CAM at critical times of development.
Purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro.
Authors: De B P , Thornton G B , Luk D , Banerjee A K
Published: May 2006

One of the major structural proteins of vesicular stomatitis virus is a small, nonglycosylated, matrix protein which associates with the nucleocapsid core during final stages of morphogenesis and budding. Biochemical and genetic studies suggested that the matrix protein regulates RNA synthesis both in vitro and in vivo. We have purified biologically active matrix protein from the virus and have directly shown that it significantly inhibits RNA synthesis in vitro mediated by the virion-associated RNA polymerase at low ionic strength (0.02 M). The inhibition was greater than 80% when the ratio of matrix protein to the major nucleocapsid protein in the transcribing complex was 2:1 (wt/wt). The inhibition was found to be at the level of RNA chain elongation and not at the initiation step. Electron microscopic studies revealed that inhibition of transcription by matrix protein was accompanied by a profound structural change of the transcribing nucleocapsid from an extended structure to a highly compact form. At higher ionic strength (0.12 M), the matrix protein failed to interact with the nucleocapsid. The matrix protein appears to be involved in condensing the nucleocapsid and blocking transcription during maturation of the virus particle.
Conformational implications of enzymatic proline hydroxylation in collagen.
Authors: Chopra R K , Ananthanarayanan V S
Published: May 2006

In 1979 it was proposed that prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) recognizes the beta-turn conformation in nascent procollagen chains and that the hydroxylation process involves a conformational change resulting in "straightening" of the beta-turn segments into the linear triple-helical conformation of native collagen. We present experimental data that verify both these postulates. The following peptides were synthesized and studied for their conformation and interaction with prolyl hydroxylase: tBoc-Pro-Gly-Ala-OH, tBoc-Pro-Gly-Val-OH, tBoc-Gly-Val-Pro-Gly-Val-OH, and tBoc-Pro-DAla-Ala-OH. Spectral data showed that these peptides preferred a beta-turn conformation. All of them acted as inhibitors of the enzyme; the pentapeptide also acted as a substrate. To mimic the biosynthetic event, a collagen model polypeptide, (Pro-Pro-Gly)10, was incubated at 37 degrees C with purified prolyl hydroxylase and the necessary cosubstrates and cofactors at pH 7.8. A progressive change from the initially nonhelical to the triple-helical conformation, as monitored by CD spectra and gel filtration, occurred during the course of proline hydroxylation. In addition to leading to increased thermal stability of the triple-helical conformation in (Pro-Pro-Gly)10 and (Pro-Pro-Gly)5, the enzymatic incorporation of the hydroxyproline residues was found to enable these polypeptides to fold into this conformation faster than the unhydroxylated counterparts. These conformational implications of proline hydroxylation in collagen may also be of use in the study of the complement subcomponent Clq and of acetylcholine esterase which contain collagen-like regions in them.
Mutant immunoglobulin genes have repetitive DNA elements inserted into their intervening sequences.
Authors: Hawley R G , Shulman M J , Murialdo H , Gibson D M , Hozumi N
Published: May 2006

The kappa light chain genes from two mutant hybridoma cell lines defective in kappa light chain synthesis were isolated and compared to the wild-type kappa light chain gene. In each case, the mutant kappa light chain genes were found to contain repetitive DNA elements in their intervening sequences that were not present in the intervening sequences of the wild-type kappa light chain gene. These elements were found to be related to the genes of intracisternal A particles. These results suggest that the decreased production of kappa light chain in the mutant cell lines is due to the presence of the intracisternal A particle-related genes.
A transposable element that splits the promoter region inactivates a Drosophila cuticle protein gene.
Authors: Snyder M P , Kimbrell D , Hunkapiller M , Hill R , Fristrom J , Davidson N
Published: May 2006

Two mutations that affect larval cuticle protein gene expression in the 2/3 variant Drosophila melanogaster strain were investigated. We demonstrate that this strain synthesizes an electrophoretic variant, fast 2 (CPf2), of wild-type cuticle protein 2(CP2). It also lacks detectable amounts of cuticle protein 3 (CP3). The other major cuticle proteins are still present. Protein and DNA sequence analyses indicate that point mutations cause two amino acid substitutions that change the electrophoretic mobility of CPf2 relative to that of CP2. The mutation abolishing the expression of CP3 was found to be a 7.3-kilobase DNA insertion located within the T-A-T-A box region of this gene, at -31 base pairs from the mRNA start site. This DNA insertion, called H.M.S. Beagle, belongs to a conserved family of repeated DNA elements that have characteristics similar to those of previously characterized Drosophila transposable elements. H.M.S. Beagle elements are repeated approximately 50 times in the haploid genome and exhibit restriction fragment-length polymorphisms around points of insertion between Canton S, Oregon R, and 2/3 Drosophila strains. Sequence analysis indicates that H.M.S. Beagle contains 266-base-pair direct repeats at its termini and is flanked by a duplication of 4 base pairs of target DNA sequence, T-A-T-A, in the CP3 gene insertion. Thus, insertion of a transposable element into the putative promoter region of the CP3 gene is evidently responsible for inactivating CP3 gene expression.
Identification of idiotypic receptors on reovirus-specific cytolytic T cells.
Authors: Ertl H C , Greene M I , Noseworthy J H , Fields B N , Nepom J T , Spriggs D R , Finberg R W
Published: May 2006

Cytolytic T lymphocytes (Tc) specific for cells infected with reovirus type 3 were shown to lyse an uninfected B-cell hybridoma line (designated 87.92.6). This hybridoma expresses and secretes an anti-idiotypic antibody that reacts with a monoclonal antibody (termed G-5). G-5 recognizes a domain on the hemagglutinin of the reovirus that is relevant to virus tropism. The Tc cell response was H-2 restricted and could be inhibited by G-5. As shown by limiting dilution analysis, identical clones lysed reovirus-infected and anti-idiotype-bearing target cells. Tc cells induced by a variant of wild-type reovirus type 3 (immunologically selected by resistance to neutralization by G-5) were unable to recognize the anti-idiotype-bearing cells although they lysed reovirus type 3-infected tumor cells. We conclude that Tc cells must bear determinants that bind anti-idiotype molecules and, furthermore, that B cells and cytolytic T cells can share these idiotypic determinants.
Production of functional human T-T hybridomas in selection medium lacking aminopterin and thymidine.
Authors: Foung S K , Sasaki D T , Grumet F C , Engleman E G
Published: May 2006

The production of hybridomas between immunologically activated T cells and malignant T-cell lines offers a potentially unlimited source of soluble T-cell-derived products. Recently, human T-T hybrids have been described; however, their use has been hampered by slow growth and chromosomal instability due at least in part to the presence of thymidine in the traditional hypoxanthine/aminopterin/thymidine (HAT) selection medium. In this report, we describe the development of a rapidly growing hypoxanthine phosphoribosyltransferase-deficient human T-cell line designated J3R7, the use of azaserine/hypoxanthine (AH) medium as an alternative selection medium to HAT medium, and the production of functional T-T hybrids by using the J3R7 line and the AH selection technique. Hybrids selected in AH medium were 4-fold greater in number and 3-fold faster in growth rate than hybrids grown in HAT medium. No stable clones were obtained from HAT cultures whereas AH-derived hybrids could be readily cloned by the method of limiting dilution. Evidence for hybridization included (i) the presence of approximately twice the number of chromosomes in hybrids than in J3R7 cells; (ii) the presence on hybrid cells of the Leu-3a surface antigen, present on normal helper T cells but not on J3R7 cells; (iii) the expression of HLA antigens of both the normal T-cell partner and the J3R7 line; and (iv) the constitutive secretion of interleukin 2 from multiple hybrid clones but not from the J3R7 cell line. Thus far, these clones have maintained their rapid growth, chromosome number, surface phenotype, and constitutive secretion of interleukin 2 for 4 months.
Antibodies to distinct polypeptides of RNA polymerase I in sera from patients with rheumatic autoimmune disease.
Authors: Stetler D A , Rose K M , Wenger M E , Berlin C M , Jacob S T
Published: May 2006

Sera from patients with rheumatic autoimmune diseases were screened for antibodies directed against RNA polymerase I by using a solid-phase radioimmunoassay. Significant levels of the antibodies were detected in the sera of all patients with either systemic lupus erythematosus or mixed connective tissue disease and in 78% of the individuals with rheumatoid arthritis. No detectable anti-RNA polymerase I antibodies were found in the sera from healthy subjects. Individuals taking hydralazine, three of whom exhibited symptoms of drug-induced lupus, had barely detectable levels of the antibodies. Immunoglobulins obtained from sera containing anti-RNA polymerase I antibodies, as determined by the radioimmunoassay, could inhibit RNA polymerase I activity in vitro. Sera from patients with systemic lupus erythematosus contained immunoglobulins directed against the polymerase I-associated polypeptide of Mr 65,000 as well as against the polypeptides of Mr 120,000 or Mr 25,000, or both. Sera from individuals with rheumatoid arthritis reacted with the polypeptide of Mr 65,000 only. The antibodies in the sera of patients with mixed connective tissue disease were directed against the Mr 42,000 polypeptide or a combination of the Mr 65,000, 42,000, and 25,000 polypeptides. These data suggest that the production of anti-RNA polymerase I antibodies may be a unique characteristic of individuals with rheumatic autoimmune diseases and that the production of antibodies against specific polypeptides of RNA polymerase I may be indicative of the particular class of disease.
Detection and transmission in chimpanzees of hepatitis B virus-related agents formerly designated "non-A, non-B" hepatitis.
Authors: Wands J R , Lieberman H M , Muchmore E , Isselbacher K , Shafritz D A
Published: May 2006

Four chimpanzees have been infected with three different inocula containing "non-A, non-B" hepatitis virus(es). After inoculation, serial studies established the presence of antigenemia or viremia (or both) by radioimmunoassay with high-affinity monoclonal antibodies directed toward separate and distinct determinants on hepatitis B surface antigen (HBsAg) and by molecular hybridization analysis using a cloned hepatitis B virus (HBV) DNA probe. In contrast to results observed during HBV infection, elevations in serum alanine aminotransferase values indicate that liver injury preceded antigenemia by several weeks. Thus, the time between inoculation and development of antigenemia (incubation period) varied from 64 to 190 days and, in some cases, single or multiple episodes of antigenemia or viremia occurred in the absence of elevated aminotransferase levels. In this study, two chimpanzees were high-titer positive for antibodies to HBsAg (anti-HBs) from previous infection with HBV, suggesting that the antigenic composition of HBV-related virus(es) is substantially different from that of HBV, since naturally occurring anti-HBs antibodies were not protective. Demonstration of HBV-related virus(es) by the methods used in this study of experimental hepatitis infection in chimpanzees should now permit detection, isolation, and characterization of these previously elusive agents.
Preparation of brain membranes containing a single type of opioid receptor highly selective for dynorphin.
Authors: James I F , Chavkin C , Goldstein A
Published: May 2006

Opioid receptors on guinea pig brain membranes were alkylated by the naltrexone analogue beta-chlornaltrexamine. Binding of the prototypical mu and kappa ligands, [3H]dihydromorphine and [3H]ethylketocyclazocine, was more readily affected by the reagent than was binding of the delta ligand, 3H-labeled [D-Ala2, D-Leu5]enkephalin. Treatment of membranes with beta-chlornaltrexamine in the presence of dynorphin resulted in significant protection of [3H]ethylketocyclazocine binding sites, without protection of [3H]dihydromorphine or 3H-labeled [D-Ala2, D-Leu5]enkephalin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanil selectively protected binding sites for 3H-labeled [D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scatchard analysis of [3H]ethylketocyclazocine binding to untreated membranes suggested two types of binding site with 40-fold difference in affinities. Membranes treated with beta-chlornaltrexamine in the presence of dynorphin retained about 40% of the high-affinity sites and lost the low-affinity sites. Selective protection of sites with high affinity for dynorphin and ethylketocyclazocine was confirmed in competition binding assays. These results strongly suggest that the three types of opioid receptor are not interconvertible and provide further evidence that the endogenous peptide dynorphin is a highly selective ligand of the kappa opioid receptor.
Genetic fine-structure mapping in human chromosome 11 by use of repetitive DNA sequences.
Authors: Gusella J F , Jones C , Kao F T , Housman D , Puck T T
Published: May 2006

A method is described for mapping of the DNA fragments of a human chromosome produced by restriction enzyme treatment of the total DNA from a hybrid cell containing a single human chromosome. The method involves production and selection of somatic cell mutants containing deletions of the human chromosome and application of a hybridization probe consisting of an individual member copy of a repetitive human DNA family. A linear map has been constructed of 19 marker DNA fragments and 5 immunological and biochemical markers on human chromosome 11, selected as a model chromosome for these studies. This approach appears to be widely applicable, is independent of cytogenetic analysis, promises to be capable of revealing the existence of rearrangements as well as deletions, appears to be amenable to further increase in resolving power, and offers potential application in various human genetic problems.
Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes.
Authors: Kolodrubetz D , Rykowski M C , Grunstein M
Published: May 2006

The yeast Saccharomyces cerevisiae contains two primary sequence subtypes of histone H2B (H2B1 and H2B2) and of H2A (H2A1 and H2A2). Mutants in each of the H2B subtypes have been used to show previously that yeast cells lacking one or the other, but not both, of the H2B proteins are viable. Because H2A protein interacts in the nucleosome with H2B, we wished to determine whether specific H2A subtypes must interact with specific H2B subtypes. We describe experiments in which frameshift mutations were introduced into both of the H2A genes in vitro and the mutant genes integrated into the yeast genome, replacing the wild-type H2A genes by a subsequent recombination. Using these mutant (hta1- and hta2-) strains we find that neither H2A gene has a unique essential function during any phase of the yeast life cycle, although strains homozygous for hta1- grow more slowly. However, one functional H2A gene is required for viability because cells mutant in both H2A genes arrest at spore germination prior to bud separation. By combining these H2A mutations with the H2B mutations obtained previously, we show that all combinations of H2A and H2B subtypes produce viable cells. From these genetic experiments and electrophoretic analysis of the histone proteins of these mutants we conclude that the H2A subtypes can associate interchangeably with the H2B subtypes.
Cloned ural locus of Schizosaccharomyces pombe propagates autonomously in this yeast assuming a polymeric form.
Authors: Sakaguchi J , Yamamoto M
Published: May 2006

DNA segments cloned from Schizosaccharomyces pombe by the ability to complement Escherichia coli pyrB mutations are shown to complement a ural mutation in S. pombe, thereby demonstrating that ural is the structural gene for aspartate transcarbamylase of S. pombe. Further, such segments combined with parts or all of pBR322 are shown to be capable of autonomous propagation in S. pombe. This suggests the existence of an autonomously replicating sequence (ars) in the vicinity of ural. Unlike the TRP1 segment cloned from Saccharomyces cerevisiae [Struhl, K., Stinchcomb, D. T., Scherer, S. & Davis, R. W. (1979) Proc. Natl. Acad. Sci. USA 76, 1035-1039], plasmids carrying the ural locus do not multiply as monomers but assume a polymeric form as large as a decamer to an icosamer in the yeast. Monomers are tandemly arranged in the polymer. Inversion of an inserted fragment or insertion of another segment into a competent plasmid greatly decreases the efficiency of such transformation, implying a role of the tertiary structure of the plasmids in the establishment of transformation of this kind.
Isolation, purification, and characterization of mouse placental lactogen.
Authors: Colosi P , Marr G , Lopez J , Haro L , Ogren L , Talamantes F
Published: May 2006

Mouse placental lactogen was purified 1840-fold from BALB/c placentae from days 14-18 of gestation with an overall yield of 29%. The purification procedure included alkaline homogenization and extraction, ammonium sulfate precipitation, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on CM- and DEAE-cellulose, and gel exclusion chromatography on Sephadex G-100. On 10% alkaline polyacrylamide gels, mouse placental lactogen had an Rf of 0.19. Electrophoresis in gels containing NaDodSO4 showed a single band with a mobility corresponding to a Mr of 23,000 +/- 1000. The isoelectric point, determined by isoelectric focusing in 8 M urea/5% 2-mercaptoethanol, was 7.1. When tested in the pigeon crop sac assay, 10 micrograms of mouse placental lactogen produced stimulation comparable with that evoked by 10 micrograms of ovine prolactin. In the rabbit mammary gland radioreceptor assay, mouse placental lactogen was 150% more potent than ovine prolactin in displacing 125I-labeled ovine prolactin from rabbit mammary gland membranes. Iodinated purified mouse placental lactogen could be displaced from rabbit mammary gland membranes by mouse placental lactogen, mouse prolactin, and ovine prolactin. Ovine prolactin was 45% as avid as mouse placental lactogen is displacing 125I-labeled mouse placental lactogen from rabbit mammary gland membranes. Mouse placental lactogen did not crossreact with antisera to mouse prolactin or mouse growth hormone in a radioimmunoassay.
Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.
Authors: Soreq H , Parvari R , Silman I
Published: May 2006

A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.
Interferon inhibits Sendai virus-induced cell fusion: an effect on cell membrane fluidity.
Authors: Chatterjee S , Cheung H C , Hunter E
Published: May 2006

Interferon can affect several cellular functions, in addition to its antiviral activity. We report here that pretreatment of human cells with homologous interferon significantly inhibits cell fusion induced by Sendai virus and that this refractory state is accompanied by a decrease in cell plasma membrane fluidity. Multinucleate cell formation induced by beta-propiolactone-inactivated Sendai virus in human fibroblast cells (a system in which fusion results from an interaction of the viral glycoprotein with the cell membrane) was inhibited by more than 90% after addition of human interferon for 18-24 hr. This inhibition could be neutralized by antiserum to interferon. Furthermore, inhibitor studies with cycloheximide and actinomycin D clearly indicated that synthesis of protein and RNA is necessary to establish the resistant state. To determine whether the inhibition of Sendai virus-induced cell fusion resulted from interferon-induced changes at the cell plasma membrane, experiments were carried out using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene, which is capable of sensing molecular motions in the hydrocarbon core of the bilayer structure. A significant decrease in the membrane fluidity of interferon-treated cells was observed. It is likely, therefore, that the inhibitory effect on Sendai virus-induced cell fusion observed in interferon-treated cells results from an increased rigidity of the target cell membrane.