bluuebunny/crossref_metadata_2025 huggingface dataset:
A promoterless retroviral vector indicates that there are sequences in U3 required for 3' RNA processing.
Authors: Dougherty J P , Temin H M
Published: May 2006

We constructed a retrovirus vector in which all viral transcriptional signals are deleted during provirus formation. This vector would be safer and more useful in gene-transfer experiments. Construction of this vector involved the deletion of all of the U3 sequence except for 10 base pairs, required for integration, from the right-side long terminal repeat. We found that this deletion resulted in an inability to propagate virus efficiently. When we inserted sequences containing all of the signals for polyadenylylation of simian virus 40 late mRNA at the end of our vector, we were able to propagate virus efficiently. This result indicates that there are sequences in U3 upstream from AAUAAA that are essential for 3' processing of viral RNA.
Transformation of normal rat kidney (NRK) cells by an infectious retrovirus carrying a synthetic rat type alpha transforming growth factor gene.
Authors: Watanabe S , Lazar E , Sporn M B
Published: May 2006

We synthesized a gene for rat type alpha transforming growth factor (TGF-alpha) consisting of the leader sequence and the sequence coding for the mature 50-amino acid peptide without the C-terminal processed region. This gene was inserted into the retrovirus vector pSW272, which is derived from spleen necrosis virus, to obtain an infectious recombinant virus carrying the rat TGF-alpha gene. This recombinant virus can infect normal rat kidney (NRK) cells and allow these cells to grow in soft agar in the presence of TGF-beta. Transformed cells isolated from colonies grown in soft agar contain an integrated form of the recombinant virus and secrete biologically active TGF-alpha into their medium. These results show that a biologically active TGF-alpha can be produced by a gene synthesized from only part of the coding region and that the infectious retrovirus carrying the TGF-alpha gene (SW355) can function as a transforming agent in NRK cells in the presence of TGF-beta.
Insulin binding to human B lymphoblasts is a function of HLA haplotype.
Authors: Kittur D , Shimizu Y , DeMars R , Edidin M
Published: May 2006

A variety of genetic and biochemical evidence points to an association between major histocompatibility complex (MHC) haplotype and several types of cell surface receptors including epidermal growth factor and insulin receptors. We report evidence for such associations between human class I MHC antigens, HLA antigens, and specific insulin binding sites on human B lymphoblasts. We have measured insulin binding to cells of an HLA-heterozygous, Epstein-Barr virus-transformed B-cell line, LCL 721, and to derivative mutants from which all or part of the HLA complex had been deleted. The affinity, Ka, of insulin binding sites is approximately 10(8) M-1 in mutants expressing antigen HLA-B5 together with other HLA antigens and in mutants expressing only HLA-C. HLA-A1; HLA-A1,B8; HLA-A2,C; and HLA null mutants (not expressing any HLA antigens) bind insulin to sites with an affinity of approximately 10(9) M-1.
Cloning of rat hepatic lipase cDNA: evidence for a lipase gene family.
Authors: Komaromy M C , Schotz M C
Published: May 2006

Clones for rat hepatic lipase were isolated by probing a rat liver cDNA library in lambda gt11 with an oligonucleotide synthesized on the basis of a partial peptide sequence. The cloned messenger codes for a protein of 472 amino acids plus a hydrophobic leader sequence of 22 amino acids. The unglycosylated protein has a predicted molecular weight of 53,222 and contains two potential sites for N-glycosylation. The protein bears striking regions of homology with other known lipases and contains peptide sequences that have been implicated in lipid binding. The homologous mRNA is present in liver tissue but no detectable mRNA is observed in the adrenal gland, despite the reported presence of hepatic lipase in both the liver and the adrenal gland. No mRNA was seen in any of a variety of other tissues.
Z-DNA in transcriptionally active chromosomes.
Authors: Lancillotti F , Lopez M C , Arias P , Alonso C
Published: May 2006

Due to the striking correlation between the distribution of transcriptionally active subdivisions of the polytene chromosomes and Z-DNA, we have addressed the question of whether the Z-DNA configuration exists in native, transcriptionally active chromosomes of Drosophila hydei prepared without interference by procedures known to induce the B to Z conformation. Our experiments indicate that Z-DNA forms are present in a specific set of sites on the native chromosomes. They occur on interbands and other subdivisions of dispersed DNA, but there is no correlation between the amount of Z-DNA detected and DNA compaction. The results suggest, moreover, that Z-DNA forms are restricted to specific genes, because various subdivisions induced to transcription in puffs show different patterns of Z-DNA. We show, in addition, that removal of chromosomal proteins by proteinase K has a strong influence on the level of anti-Z-DNA reactivity.
Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.
Authors: Hornsby P J , Hancock J P , Vo T P , Nason L M , Ryan R F , McAllister J M
Published: May 2006

Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17 alpha-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17 alpha-hydroxylase enzyme activity. The lower levels of 17 alpha-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells results from a general decline in response to several growth factors. However, the decline in 17 alpha-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17 alpha-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17 alpha-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17 alpha-hydroxylase. The loss of 17 alpha-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17 alpha-hydroxylase, because adrenocortical cell clones that had high levels of 17 alpha-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17 alpha-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17 alpha-hydroxylase mRNA after incubation with the inducing agent.
Structure of a cytotoxic T-lymphocyte-specific gene shows a strong homology to fibrinogen beta and gamma chains.
Authors: Koyama T , Hall L R , Haser W G , Tonegawa S , Saito H
Published: May 2006

Using a subtractive cDNA cloning method, we isolated a number of T-lymphocyte-specific genes. One of these genes, represented by the cDNA clone pT49, is expressed in cytotoxic T lymphocytes but not in helper T lymphocytes or B lymphocytes. The protein structure deduced from the nucleotide sequence showed a high degree of homology to fibrinogen beta and gamma subunits. This might indicate that evolutionarily fibrinogen has its origin in lymphocytes. In spite of the strong homology of pT49 protein to the fibrinogen subunits, the positions of the introns in the pT49 gene are totally different from those of the fibrinogen genes.
Peptide and nucleotide sequences of rat CD4 (W3/25) antigen: evidence for derivation from a structure with four immunoglobulin-related domains.
Authors: Clark S J , Jefferies W A , Barclay A N , Gagnon J , Williams A F
Published: May 2006

The rat W3/25 antigen was the first marker antigen of helper T lymphocytes to be identified. Subsequently, the human OKT4 antigen (now called CD4) was described, and cell distribution and functional data suggested that W3/25 and OKT4 antigens were homologous. This is now confirmed by the matching of peptide sequences from W3/25 antigen with sequence predicted from rat cDNA clones detected by cross-hybridization with a cDNA probe for human CD4. Analysis of the two sequences suggests an evolutionary origin from a structure with four immunoglobulin-related domains, although only domain 1 at the NH2 terminus meets the standard criteria for an immunoglobulin-related sequence. CD4 domains 2 and 4 contain disulfide bonds but seem like truncated immunoglobulin domains, whereas domain 3 may have a pattern of beta-strands like an immunoglobulin variable domain, but without the disulfide bond.
Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen.
Authors: Husson R N , Young R A
Published: May 2006

Mycobacterium tuberculosis genes encoding immunologically relevant proteins were isolated by systematically screening a lambda gt11 recombinant DNA expression library with a collection of murine monoclonal antibodies directed against protein antigens of this pathogen. These antibodies, previously characterized by a World Health Organization workshop on monoclonal antibodies against mycobacteria, were used to isolate DNA sequences encoding five major protein antigens of this pathogen. To evaluate the extent of crossreactivity between these M. tuberculosis antigens and antigens of Mycobacterium leprae, recombinant antigens were probed with monoclonal antibodies directed against the protein antigens of these bacilli. One of the antigens, a 65-kDa protein, has determinants common to M. tuberculosis and M. leprae. We find not only that this antigen is recognized by mouse monoclonal antibodies but that it is the major protein recognized by anti-M. tuberculosis rabbit sera. The 65-kDa proteins of M. tuberculosis and M. leprae appear to play a role in the humoral and cell-mediated immune response to these pathogens.
The suprachiasmatic nuclei contain a tetrodotoxin-resistant circadian pacemaker.
Authors: Schwartz W J , Gross R A , Morton M T
Published: May 2006

Tetrodotoxin was infused into the suprachiasmatic nuclei of unanesthetized and unrestrained rats continuously for 14 days. The internal timekeeping mechanism of the circadian pacemaker in the nuclei continued to oscillate unaffected by this treatment, although the toxin reversibly blocked function of both the input pathway for pacemaker entrainment and an output pathway for expression of the circadian drinking rhythm. Thus, Na+-dependent action potentials appear necessary for entrainment and expression of overt circadian rhythms, but they do not seem necessary for the pacemaker to keep accurate time. The experimental approach presented in this paper is useful because it allows systematic assessment and distinction of the input, pacemaker, and output components of a mammalian circadian timekeeping system in vivo.
Molecular cloning and sequencing of a cDNA for olfactory marker protein.
Authors: Rogers K E , Dasgupta P , Gubler U , Grillo M , Khew-Goodall Y S , Margolis F L
Published: May 2006

cDNA clones corresponding to mRNA for rat olfactory marker protein (OMP) were isolated from a cDNA library. The library was constructed from olfactory mucosa poly(A)+ RNA enriched for OMP mRNA and cloned into a pBR322-derived plasmid, pMG5. OMP cDNA clones were detected by using a 17-base oligonucleotide probe that contained all 16 possible sequences coding for a known partial amino acid sequence of rat OMP. The identity of these clones was confirmed by hybrid-selected translation and nucleotide sequencing. The sequence of one clone was determined and contained the complete OMP coding region of 486 nucleotides followed by 1630 nucleotides of the 3' untranslated region. The 3' untranslated region included the polyadenylylation signal 16 nucleotides upstream of the poly(A) tail. No other ATG-initiated open reading frame larger than 20 codons was present in register. RNA blot analysis of olfactory mucosa poly(A)+ RNA using this clone as a probe indicated that the level of OMP mRNA, but not its size, declined significantly within a few days following olfactory bulbectomy. OMP mRNA was not detected in 14 nonolfactory rat tissues. Surprisingly, a small amount of OMP mRNA was observed in olfactory bulb. The presence of OMP mRNA in olfactory bulb was confirmed by in vitro translation and immunoprecipitation. These results suggest either that a previously undescribed population of neurons in the olfactory bulb synthesize OMP or that OMP mRNA is transported to the bulb by axonal transport.
Role of sialic acid in synaptosomal transport of amino acid transmitters.
Authors: Zaleska M M , Erecińska M
Published: May 2006

Active, high-affinity, sodium-dependent uptake of gamma-aminobutyric acid and of the acidic amino acid D-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the gamma-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake.
Insulin-activated tyrosine phosphorylation of a 15-kilodalton protein in intact 3T3-L1 adipocytes.
Authors: Bernier M , Laird D M , Lane M D
Published: May 2006

Insulin stimulates phosphorylation of a tyrosine residue(s) on a 15-kDa protein (p15), and the cytosolic phosphorylated protein (pp15) accumulates only when 3T3-L1 adipocytes are treated with phenylarsine oxide. It has been shown previously that phenylarsine oxide, an agent that complexes vicinal dithiols, interrupts signal transmission from the insulin receptor to the glucose transport system. Several lines of evidence presented here indicate the involvement of pp15 in insulin receptor-initiated signal transduction to the glucose transport system. The reciprocal effects of phenylarsine oxide on the insulin-activated accumulation of pp15 and on insulin-stimulated hexose uptake are reversed by the vicinal dithiol 2,3-dimercaptopropanol but not by the monothiol 2-mercaptoethanol. Thus, a cellular dithiol appears to function in the signal transmission pathway downstream from pp15. Like the insulin-activated autophosphorylation of the receptor's beta subunit (on tyrosine), activation of phosphorylation of p15 is specific, with insulin-like growth factors 1 and 2, epidermal growth factor, and platelet-derived growth factor being inactive. Moreover, both processes exhibit identical insulin concentration dependence. The temporal kinetic relationship of insulin-activated receptor beta-subunit phosphorylation, followed by the phosphorylation of p15 and then increased hexose uptake rate, is consistent with an intermediary signaling role for pp15 in insulin-stimulated glucose uptake.
Optional exon in the 5'-untranslated region of 3-hydroxy-3-methylglutaryl coenzyme A synthase gene: conserved sequence and splicing pattern in humans and hamsters.
Authors: Gil G , Smith J R , Goldstein J L , Brown M S
Published: May 2006

3-Hydroxy-3-methylglutaryl coenzyme A synthase (hydroxymethylglutaryl-CoA synthase, EC 4.1.3.5) is a negatively regulated enzyme in the synthetic pathway for cholesterol, isopentenyl tRNA, and other isoprenoids. The 5'-untranslated region of the mRNA for Chinese hamster hydroxymethylglutaryl-CoA synthase contains an optional exon of 59 nucleotides located 10 nucleotides upstream of the translation start site. About 50% of the mRNAs contain this exon, and the other 50% lack it owing to differential intron splicing. We show that the two transcripts are found in similar ratios in multiple tissues of the Syrian hamster, including the brain. The relative amounts of the two transcripts in brain and liver are constant from day 0 to day 75 of life. A similar alternative splicing pattern for hydroxymethylglutaryl-CoA synthase was observed in three human tissues: cultured fibroblasts, fetal adrenal gland, and fetal liver. A cDNA for human synthase had 90% homology to the hamster sequence in the region corresponding to the optional exon. This sequence contains a 20 out of 26 nucleotide match with the sequence immediately upstream of the initiator AUG codon in the mRNA for hamster hydroxymethylglutaryl-CoA reductase, the enzyme that follows the synthase in the isoprenoid biosynthetic pathway. These findings raise the possibility that the optional exon plays an important, conserved functional role in humans and hamsters.
Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells.
Authors: Jay D G , Gilbert W
Published: May 2006

DNA can be encapsulated into lipid vesicles formed by sonication. The presence of a basic protein, lysozyme, enhances the incorporation 100-fold above the level expected by random trapping. This is demonstrated by the ability of the lipid vesicles to protect DNA from digestion with DNase. Such an enhancement of nuclei acid incorporation into vesicles by basic polypeptides and the sharply increased concentration of these macromolecules in the internal volume may have been advantageous in prebiotic evolution.
Mapping the X-linked lymphoproliferative syndrome.
Authors: Skare J C , Milunsky A , Byron K S , Sullivan J L
Published: May 2006

The X-linked lymphoproliferative syndrome is triggered by Epstein-Barr virus infection and results in fatal mononucleosis, immunodeficiency, and lymphoproliferative disorders. This study shows that the mutation responsible for X-linked lymphoproliferative syndrome is genetically linked to a restriction fragment length polymorphism detected with the DXS42 probe (from Xq24-q27). The most likely recombination frequency between the loci is 4%, and the associated logarithm of the odds is 5.26. Haplotype analysis using flanking restriction fragment length polymorphism markers indicates that the locus for X-linked lymphoproliferative syndrome is distal to probe DXS42 but proximal to probe DXS99 (from Xq26-q27). It is now possible to predict which members of a family with X-linked lymphoproliferative syndrome are carrier females and to diagnose the syndrome prenatally.
Acetylcholine increases voltage-activated Ca2+ current in freshly dissociated smooth muscle cells.
Authors: Clapp L H , Vivaudou M B , Walsh J V , Singer J J
Published: May 2006

The regulation of voltage-activated Ca2+ current by acetylcholine was studied in single freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus by using the tight-seal whole-cell recording technique. Ca2+ currents were elicited by positive-going command pulses from a holding level near -80 mV in the presence of internal Cs+ to block outward K+ currents. Ca2+ current was greatest in magnitude at command potentials near 10 mV. At such command potentials, acetylcholine increased the magnitude of the inward current and slowed its decay. The effects of acetylcholine were seen in the absence of external Na+ or with low Cl- (aspartate replacement) in the bathing solution and could be mimicked by muscarine. The peak of the current-voltage relationship for the Ca2+ current was not discernibly shifted along the voltage axis by acetylcholine. These results demonstrate that activation of muscarinic receptors not only suppresses a K+ current (M-current), as we have previously demonstrated [Sims, S. M., Singer, J. J. & Walsh, J. V., Jr. (1985) J. Physiol. (London) 367, 503-529], but also increases the magnitude and slows the decay of Ca2+ current.
Expression of the hepatitis B virus X gene in mammalian cells.
Authors: Siddiqui A , Jameel S , Mapoles J
Published: May 2006

The hepatitis B virus (HBV) DNA contains an open reading frame (ORF) designated X, which has the capacity to encode a protein of 16,560 Da (subtype adw). Such a protein has not been identified in either HBV particles or infected human livers, and therefore its role in the viral life cycle remains unknown. We report here the expression of the HBV X ORF in cultured cells using recombinant vectors. A protein of 16 kDa was identified by means of an antiserum prepared against a synthetic peptide and with human antisera from hepatitis B patients as well as those with hepatocellular carcinoma. Cell fractionation and immunofluorescence studies suggest a probable association with cytoskeletal components. Our studies further located a promoter sequence upstream of the X ORF, which directs the transcription of a 0.7- to 0.8-kilobase X-specific RNA in transfected human hepatoma cells.
Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization.
Authors: Armstrong D , Eckert R
Published: May 2006

Two classes of calcium channels were activated by membrane depolarization in cell-free membrane patches from GH3 cells, an electrically excitable cell line derived from a mammalian pituitary tumor. One class had a conductance of approximately 10 pS in 90 mM barium, had a threshold of activation near -40 mV, and was inactivated rapidly at holding potentials more positive than -80 mV. The other class, with a conductance of approximately 23 pS and a threshold nearer -20 mV, did not inactivate in barium but stopped responding to depolarization altogether when the cytoplasmic side of the patch was exposed to a standard physiological saline solution. Buffering the concentration of calcium ions to less than 10 nM on the cytoplasmic side did not prevent this loss of activity. However, activity was restored and maintained for the duration of the patch when the catalytic subunit of cAMP-dependent protein kinase was added with MgATP to the cytoplasmic side of the membrane. Cell-free patch formation in the presence of the dihydropyridine, BAY K 8644, also delayed the loss of activity, but unlike the catalytic subunit plus ATP, BAY K 8644 alone did not restore activity when it was added after the channels no longer responded to depolarization. Evidently the dihydropyridine-sensitive class of voltage-activated calcium channels must be phosphorylated in order to open when the membrane is depolarized. That hypothesis provides a simple framework for understanding the modulation of calcium channel gating by neurotransmitters, calcium ions, and dihydropyridines.
Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions.
Authors: Vandenberg C A
Published: May 2006

The mechanism of rectification of the inwardly rectifying potassium channel was examined with single-channel recording techniques in isolated ventricular myocytes from adult guinea pig heart. Inward, or anomalous, rectification describes the property that potassium (K) current can enter the cell at potentials negative to the potassium equilibrium potential, EK, more readily than it can leave the cell at positive potentials. Voltage ramps applied to single inward rectifier channels in cell-attached patches produced single-channel currents that rectified strongly with a marked reduction in current at a potential near EK. At more positive potentials no current could be detected. Rectification was influenced by external and internal K concentrations. Single-channel activity, which usually disappears rapidly in excised patches, could be maintained by removing calcium from the internal solution. Rectification could be eliminated by excision of the patch into an internal solution in which free magnesium (Mg2+) was reduced to less than 1 microM, and it could be restored by the addition of approximately 1 mM Mg2+ to the internal solution. At intermediate concentrations of Mg2+, intermediate degrees of rectification were obtained, and the current at potentials positive to EK was often interrupted by brief closures. These studies suggest that rectification is due to internal block by Mg2+, possibly the result of rapid block of the open channel.
Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.
Authors: Hediger M A , Ikeda T , Coady M , Gundersen C B , Wright E M
Published: May 2006

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl alpha-D-[14C]glucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (Vmax) of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/glucose cotransporter was found in a single fraction of approximately 2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.
Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action.
Authors: Fox J A , Soliz N M , Saltiel A R
Published: May 2006

Insulin stimulates the hydrolysis of a phosphatidylinositol-glycan, resulting in the generation of two related inositol phosphate-glycan enzyme modulators and diacylglycerol. A phosphatidylinositol-glycan-specific phospholipase C that catalyzed this reaction was found in the plasma-membrane fraction of rat liver. The enzymatic activity was measured by the release of diacylglycerol from the glycosylated-phosphatidylinositol-membrane-anchored variant surface glycoproteins of African trypanosomes. The enzyme also catalyzed the production of an inositol phosphate-glycan from an insulin-sensitive phosphatidylinositol-glycan precursor. The enzyme was solubilized with neutral nonionic detergent and purified to near homogeneity by anion-exchange chromatography on DEAE-cellulose, followed by hydrophobic chromatography on butyl-agarose. The resulting enzyme preparation was purified approximately 20,000-fold from whole liver and exhibited one major silver-stained band of Mr 52,000 on NaDodSO4/PAGE. Gel permeation chromatography of the purified activity revealed a Stokes radius of 35 A and an apparent molecular weight of 62,000, suggesting that the enzyme was tightly associated with lipid or detergent but existed as a monomer in its active form. The enzyme was specific for glycosylated phosphatidylinositol; no hydrolysis of phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate, or phosphatidylcholine was observed. The enzyme was calcium independent and thiol activated. These data suggest a role for the phosphatidylinositol-glycan-specific phospholipase C as an effector for some of the metabolic actions of insulin.
Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts.
Authors: Ober S S , Pardee A B
Published: May 2006

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pHi) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. This increase of pHi in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na+/H+-antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pHi regulation in the tumorigenic CHEF/18 derivatives lies in the Na+/H+ antiporter itself. Further studies to determine the biological significance of an increased pHi show that the external pH (pHo)-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pHi in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pHi can contribute to the acquisition of altered growth properties.
Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule, L-CAM.
Authors: Gallin W J , Sorkin B C , Edelman G M , Cunningham B A
Published: May 2006

The liver cell adhesion molecule (L-CAM) appears on non-neural epithelial tissues and mediates calcium-dependent adhesion in these tissues both in the embryo and in the adult. It appears on cell surfaces as a glycoprotein of Mr 124,000 but is synthesized as a precursor of Mr 135,000. We have isolated and determined the nucleic acid sequence of a cDNA clone (lambda L320) encoding chicken L-CAM. The 5' end of this clone has an open reading frame extending for 2520 base pairs, followed by an 850-base-pair untranslated region terminating with a polyadenylylation site at its 3' end. Protein sequence analysis of intact L-CAM and of cyanogen bromide fragments of the protein confirmed the reading frame and indicated that lambda L320 encodes the complete sequence of L-CAM as it is expressed on the cell surface as well as the bulk of the precursor. The sequence includes a hydrophobic segment of 31 amino acids, supporting our earlier conclusion that L-CAM is an intrinsic membrane protein. There are five potential asparagine glycosylation sites on the extracellular part of the molecule and an intracellular domain that is phosphorylated in vivo. The mature L-CAM polypeptide consists of 727 amino acids, with a calculated Mr of 79,900 for the carbohydrate-free protein. The L-CAM sequence is not homologous to other known protein sequences, including those of the neural cell adhesion molecule (N-CAM) and other members of the immunoglobulin superfamily, but the L-CAM molecule does contain three contiguous segments (113 amino acids each) that are homologous to each other. The similarities among these segments suggest that at least part of the L-CAM molecule arose by gene duplication.
Cloning and characterization of a dispersed, multicopy, X chromosome sequence in Drosophila melanogaster.
Authors: Waring G L , Pollack J C
Published: May 2006

We have isolated and characterized a dispersed middle repetitive DNA sequence from Drosophila melanogaster that is concentrated on the euchromatic portion of the X chromosome. In situ hybridization of the repeat unit to salivary gland chromosomes shows the sequence is distributed among approximately 10 major and 20 minor X chromosomal sites. Based on DNA sequence analysis of homologous sequences from three different cytogenetic regions, the 372-base-pair repeat unit appears to be (A + T)-rich and noncoding and shows strong sequence conservation among units from different chromosomal regions. The nature and distribution of this sequence are suggestive of the hypothetical X chromosome DNA sequences thought to be involved in the primary establishment of sex determination and dosage compensation in Drosophila.
Presence of antibodies against a cell-surface protein, cross-reactive with DNA, in systemic lupus erythematosus: a marker of the disease.
Authors: Jacob L , Lety M A , Choquette D , Viard J P , Jacob F , Louvard D , Bach J F
Published: May 2006

Antibodies against a cell-surface protein, cross-reactive with double-stranded DNA, were detected in the serum of 25 patients with active human systemic lupus erythematosus (SLE), defined on the basis of the revised American Rheumatism Association classification. Among these sera, two did not display anti-DNA antibodies, as shown by Farr assay, solid-phase radioimmunoassay, and Crithidia luciliae test. Five other SLE patients were consecutively studied in active and remission states. Antibodies against the protein were detected in the serum of the 5 SLE patients when they were in active phase but not in the serum of the same patients in inactive phase of the disease. The anti-protein antibodies were not found in the serum of 10 inactive SLE patients or in the sera of 10 normal human controls, 10 patients with rheumatoid arthritis, 5 patients with scleroderma, and 4 patients with primary sicca syndrome. Taken together, these results strongly suggest that antibodies against this cell-surface protein could provide a better diagnosis marker and activity index than anti-DNA antibodies in SLE.
Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts.
Authors: Mattes P M , Maloney P C , Littlefield J W
Published: May 2006

An abnormal regulation of chloride permeability has been described for epithelial cells from patients with cystic fibrosis (CF). To learn more about the biochemical basis of this inherited disease, we have studied chloride metabolism in cultured CF fibroblasts by comparing the efflux of 36Cl- from matched pairs of CF and normal fibroblasts. The rate constants describing 36Cl- efflux did not differ between the two cell types, but in each of the four pairs tested the amount of 36Cl- contained within CF cells was consistently reduced, by 25-30%, relative to normal cells. Comparisons of cell water content and 22Na+ efflux showed no differences between the two cell types, suggesting that overall intracellular chloride concentration is lower than normal in CF fibroblasts. Such data suggest that the CF gene defect is expressed in skin fibroblasts and that this defect may alter the regulation of intracellular Cl- concentration, perhaps through changes in Cl- permeability.
Stark effect spectroscopy of
Rhodobacter sphaeroides
and
Rhodopseudomonas viridis
reaction centers
Authors: Lockhart David J. , Boxer Steven G.
Published: May 2006

The nature of the initially excited state of the primary electron donor or special pair has been investigated by Stark effect spectroscopy for reaction centers from the photosynthetic bacteria
Rhodopseudomonas viridis
and
Rhodobacter sphaeroides
at 77 K. The data provide values for the magnitude of the difference in permanent dipole moment between the ground and excited state, [unk]Δμ[unk], and the angle [unk] between Δμ and the transition dipole moment for the electronic transition. [unk]Δμ[unk] and [unk] for the lowest-energy singlet electronic transition associated with the special pair primary electron donor were found to be very similar for the two species. [unk]Δμ[unk] for this transition is substantially larger than for the Q
y
transitions of the monomeric pigments in the reaction center or for pure monomeric bacteriochlorophylls, for which Stark data are also reported. We conclude that the excited state of the special pair has substantial charge-transfer character, and we suggest that charge separation in bacterial photosynthesis is initiated immediately upon photoexcitation of the special pair. Data for
Rhodobacter sphaeroides
between 340 and 1340 nm are presented and discussed in the context of the detection of charge-transfer states by Stark effect spectroscopy.
Accelerated autoxidation and heme loss due to instability of sickle hemoglobin.
Authors: Hebbel R P , Morgan W T , Eaton J W , Hedlund B E
Published: May 2006

The pleiotropic effect of the sickle gene suggests that factors in addition to polymerization of the mutant gene product might be involved in sickle disease pathobiology. We have examined rates of heme transfer to hemopexin from hemoglobin in dilute aqueous solution (0.5 mg of Hb per ml) at 37 degrees C. HbO2 S loses heme 1.7 times faster than HbO2 A, with apparent rate constants of 0.024 hr-1 and 0.014 hr-1, respectively. In contrast, Hb A and Hb S behave identically in their MetHb forms (very rapid heme loss) and their HbCO forms (zero heme loss). This indicates that the faster heme loss from HbO2 S is due to accelerated autoxidation (HbO2----MetHb) rather than to some other type of instability inherent in the relationship of sickle heme to its pocket in globin. This interpretation is supported by spectrophotometric measurement of initial rates of MetHb formation during incubation at 37 degrees C. This directly shows 1.7 times faster autoxidation, with apparent rate constants of 0.050 hr-1 for HbO2 S and 0.029 hr-1 for HbO2 A. While the participation of this process in the cellular pathobiology of sickle erythrocytes remains unproven, the present data are consistent with, and perhaps help explain, two prior observations: the excessive spontaneous generation of superoxide by sickle erythrocytes; and the abnormal deposition of heme and heme proteins on membranes of sickle erythrocytes.
Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions.
Authors: Meunier H , Rivier C , Evans R M , Vale W
Published: May 2006

The S1-nuclease analysis was used to investigate the pattern of inhibin expression in the rat. In a first series of experiments, expression of the alpha, beta A, and beta B subunits of inhibin were monitored in various tissues from male and female rats. Two observations emerge from these studies. First, expression of inhibin subunits was found in gonadal and extragonadal tissues. In addition to the ovary and testis, inhibin alpha, beta A, and beta B RNAs were detected in the placenta, pituitary, adrenal, bone marrow, kidney, spinal cord, and brain. Detection of inhibin RNAs in the brain and spinal cord suggested that these subunits may exert neuroregulatory functions in the central and peripheral nervous systems. Furthermore, the presence of inhibin alpha and beta subunits in the placenta and the pituitary gland, two cell types that have clearly been shown to be regulated by exogenous inhibin, may reflect existing paracrine and/or autocrine processes active in these tissues. The second observation is that expression of inhibin subunit RNAs may vary by severalfold in a tissue-specific fashion. for example, alpha-subunit RNA levels are abundant in the gonads, whereas beta A-subunit RNA is predominant in the placenta and bone marrow. Finally, it is noted that expression of testicular inhibin RNA subunits decreases during sexual maturation. We conclude that the dimers comprised of inhibin subunits possess diverse functions and may act as growth/differentiation factors as well as a hormone.
A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody.
Authors: Deutscher S L , Keene J D
Published: May 2006

An autoantibody from a patient with lupus-overlap syndrome was found to bind a specific region of U1 RNA. By using RNA sequence analysis, immunoprecipitation, and competition experiments with in vitro synthesized fragments of U1 RNA, a region of 40 nucleotides representing a stem-loop secondary structure was found to be an immunoreactive domain. This antibody recognized a conformational epitope because neither the RNA stem nor the RNA loop alone was immunoprecipitable. Antisense U1 RNA, U1 DNA, and other small RNAs were not reactive with the antibody. While the origins of nucleic acid-binding antibodies are unknown, this RNA-specific autoantibody probably originated by direct presentation to the immune system or as an anti-idiotype against a more common U1 small nuclear ribonucleoprotein-specific autoantibody. Thus, these findings have implications for the mechanisms of autoimmune recognition and provide an immunological approach to probing RNA structure and protein-RNA interactions.
Isolation and characterization of natural allene oxides: unstable intermediates in the metabolism of lipid hydroperoxides.
Authors: Brash A R , Baertschi S W , Ingram C D , Harris T M
Published: May 2006

Allene oxides are unstable epoxides that have been implicated as intermediates in the biotransformation of hydroperoxyicosatetraenoic acids and related hydroperoxides to ketols and cyclopentenones. Direct proof of the structure of the putative allene oxide intermediates has been hampered by their extreme instability under the conditions of their biosynthesis (t1/2 approximately 15-30 sec at 0 degree C and pH 7.4). We now report the isolation and structural elucidation of allene oxides prepared from the (13S)-hydroperoxides of linoleic and linolenic acids. The compounds were biosynthesized by using a very active enzyme preparation from flaxseed. After a 5-sec incubation at 0 degrees C, the allene oxide metabolites were extracted and purified as the methyl ester derivatives at -15 degrees C. The structures were established by UV, CD, NMR, and oxygen-18 labeling experiments. 12,13(S)-Oxido-9Z,11-octadecadienoic acid is derived from linoleic acid, and 12,13(S)-oxido-9Z,11,15Z-octadecatrienoic acid is from linolenic acid. Analysis of the breakdown products formed on exposure to water led to identification of hydrolysis and cyclization products previously characterized as enzymic derivatives of the (13S)-hydroperoxides in flaxseed. Our results give direct proof of the structure of the allene oxide intermediates and should facilitate further investigation of the metabolism of this class of epoxide to prostaglandins, clavulones, and other stable end products.
A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing.
Authors: Carrington J C , Dougherty W G
Published: May 2006

Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.
Cytopathic effect of human immunodeficiency virus in T4 cells is linked to the last stage of virus infection.
Authors: Leonard R , Zagury D , Desportes I , Bernard J , Zagury J F , Gallo R C
Published: May 2006

A principal feature of acquired immunodeficiency syndrome is depletion of T4 lymphocytes, which is partly due to a direct cytopathic effect of the virus. Both syncytial formation (viral-induced cell fusion) and premature cell death have been cited as the major cause for this phenomenon. By kinetic analysis of cell proliferation and cell lysis we show that the cytopathic effect correlates chiefly with virus production from infected cells, including giant syncytial cells. Most T4 cells were, at least transiently, infected by human immunodeficiency virus (human T-lymphotropic virus type IIIB strain); however, after phytohemagglutinin activation, only 10-30% of infected cells express virus (and die) at any one time, indicating that virus production, followed by cell killing, is linked to immune activation and cell differentiation. We also show that an interval exists before viral release, in which expression of viral antigens occurs on the cell surface, suggesting that infected cells are immunogenic before viral production. If so, they may induce a cell-mediated immune response that could minimize dissemination of human immunodeficiency virus, a possibility that has influenced our approaches to the development of a vaccine for prevention of acquired immunodeficiency syndrome.
ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae.
Authors: Sugino A , Nitiss J , Resnick M A
Published: May 2006

An activity that catalyzes the transfer of a strand from a duplex linear molecule of DNA to a complementary circular single strand can be detected in crude extracts from mitotic and meiotic cells of the yeast Saccharomyces cerevisiae by adding yeast single-stranded DNA binding proteins. This DNA strand-transfer activity increases greater than 15-fold during meiosis in MATa/MAT alpha diploids prior to the detection of a 100- to 1000-fold increase in homologous chromosomal recombination. No increase is observed in MATa/MATa or MAT alpha/MAT alpha cells, which do not undergo meiosis when shifted to meiotic medium, suggesting the activity is related to meiotic recombination. The activity is named strand-transfer protein alpha (STP alpha) and has been extensively purified from the meiotic cells (6 hr after exposure to sporulation medium). The apparent molecular mass of STP alpha is 38 kDa under denaturing conditions. The DNA strand-transfer reaction catalyzed by STP alpha requires homologous single-stranded and double-stranded DNA and Mg2+ but no nucleotide cofactor. Yeast single-stranded DNA binding proteins stimulate the reaction at least 10-fold. Among the products analyzed by electron microscopy were typical strand-exchange structures.
DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.
Authors: Pizzagalli A , Valsasnini P , Plevani P , Lucchini G
Published: May 2006

A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.
The possible role of ammonia in phototaxis of migrating slugs of
Dictyostelium discoideum
Authors: Bonner J. T. , Chiang A. , Lee J. , Suthers H. B.
Published: May 2006

Previously we showed that the rising cell masses of cellular slime molds orient away from high concentrations of ammonia gas, presumably by speeding up the cells on one side. Here we show that in the same way NH
3
could also be involved in the highly sensitive phototaxis found in the migrating slugs of
Dictyostelium discoideum
. We have evidence that light increases their speed of migration and their production of NH
3
. Since unilateral light is concentrated on the distal side of a cell mass by the “lens effect,” this leads to the obvious hypothesis that the light stimulates the local production of NH
3
, which, in turn, stimulates the cells in the illuminated region to move faster.