bluuebunny/crossref_metadata_2025 huggingface dataset:
Tangier disease: a disorder of intracellular membrane traffic.
Authors: Schmitz G , Assmann G , Robenek H , Brennhausen B
Published: May 2006

The interaction of human high density lipoproteins (HDL) with isolated human monocytes from control and Tangier patients was studied in tissue culture experiments. It was observed that normal monocytes, similar to mouse peritoneal macrophages, bind HDL to a cell surface receptor, internalize the bound HDL particles, transport the internalized HDL intracellularly through the cytoplasmic compartment without significant degradation, and ultimately resecrete intact HDL. The cellular interaction of Tangier monocytes with normal HDL was markedly different from control monocytes. HDL binding to Tangier monocytes was moderately increased and cell-associated HDL radioactivity was 6- to 10-fold enhanced in Tangier monocytes. The bulk of the internalized HDL, however, was detected in secondary lysosomes. Only minor amounts of the internalized HDL were resecreted from the Tangier monocytes, and most of this was degraded. These data suggest that the cellular metabolism of HDL is abnormal in Tangier monocytes. It is postulated that Tangier disease may be a disorder of intracellular membrane traffic in which HDL is diverted into the lysosomal compartment and degraded instead of being secreted through its regular transcellular route.
Replication timing of the H4 histone genes in Physarum polycephalum.
Authors: Jalouzot R , Toublan B , Wilhelm M L , Wilhelm F X
Published: May 2006

The time of replication of the two H4 histone genes (H41 and H42) was determined during the naturally synchronous mitotic cycle of Physarum polycephalum. 5-Bromo-2'-deoxyuridine labeling and density gradient centrifugation was used to isolate newly synthesized DNA from defined periods of S phase. The DNA was analyzed by Southern hybridization with a cloned probe containing one of the H4 histone genes of Physarum. The results indicate that the two H4 histone genes are replicated in the first 30 min of S phase but not exactly at the same time. H41 is replicated during the first 10 min of S phase, when only 15% of the genome is duplicated, whereas H42 replicates between 20 and 30 min after the onset of S phase. The possible relationship between the periodic expression of the genes and the timing of their replication is discussed.
The structure of caltrin, the calcium-transport inhibitor of bovine seminal plasma.
Authors: Lewis R V , Agustin J S , Kruggel W , Lardy H A
Published: May 2006

The amino acid sequence of the bovine seminal protein, caltrin, which inhibits calcium transport into spermatozoa, has been determined. The protein contains 47 amino acid residues. Parts of the sequence are identical with that reported for bovine seminal plasmin, a protein possessing antibacterial activity. We believe the proteins are identical and that the previously reported sequence of seminal plasmin is in error.
Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia.
Authors: Hitotsumachi S , Carpenter D A , Russell W L
Published: May 2006

In order to maximize the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse stem-cell spermatogonia, advantage was taken of the fact that these cells can accumulate mutations from repeated doses given over relatively long time periods. Repeated doses (100 mg/kg) of ethylnitrosourea injected intraperitoneally into male mice at weekly intervals were found to allow adequate survival and fertility with total dosages of 300 and 400 mg/kg. The specific-locus mutation frequencies at these dosages were, respectively, 1.8 and 2.2 times that obtained with the maximal practicable single dose of 250 mg/kg. The mutation frequency induced by a 400 mg/kg dosage of ethylnitrosourea is 12 times the maximal mutation frequency achievable with a single exposure to x-rays and 36 times that reported for procarbazine, the most effective chemical mutagen previously known for mouse stem-cell spermatogonia. Ethylnitrosourea is already the mutagen of choice in deliberate attempts to create mouse models for human disease and in any experiments in which a maximal mutation rate is desired. Repeated-dose regimens similar to the ones reported here should increase the efficiency of such studies.
Monoclonal antibodies against human Ia antigens stimulate monocytes to secrete interleukin 1.
Authors: Palacios R
Published: May 2006

The monoclonal antibodies (mAb) DA6.147, DA6.164, and HIG.48 against human Ia antigens, but not the W6/32 mAb against human class I major histocompatibility complex antigens or the anti-monocyte OKM1 and 63D3 mAb, stimulated monocytes to secrete interleukin 1 (IL-1). IL-1 was measured by its property of promoting the production of interleukin 2 (IL-2) by phytohemagglutinin-treated LBRM-33 clone 1A5 cells. IL-1 activity induced by anti-Ia antibodies could be detected 24 hr after initiation of the cultures and reached its highest levels at days 3-4 of culture. Concentrations of 1 microgram/ml or higher of the anti-Ia antibodies induced monocytes to secrete significant levels of IL-1 activity. The anti-Ia mAb induced Ia-bearing but not Ia-negative monocytes to secrete IL-1. Both Ia-positive and Ia-negative monocytes produced IL-1 activity under the stimulus of lipopolysaccharide. It is concluded that the DA6.147, DA6.164, and HIG.48 mAb stimulate secretion of IL-1 by interacting Ia antigens on monocytes. The data support the view that besides serving as restricting elements for recognition of foreign antigens by T cells, Ia antigens may also function as transducer elements.
Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes.
Authors: Maller J L , Foulkes J G , Erikson E , Baltimore D
Published: May 2006

Phosphorylation of ribosomal protein S6 in NIH 3T3 fibroblasts is dependent on the presence of serum, but after transformation of these cells by Abelson murine leukemia virus (Ab-MuLV), S6 remained highly phosphorylated on serine residues either in the absence or the presence of serum. To investigate whether S6 phosphorylation in this system was a consequence of the action of the Ab-MuLV tyrosine-specific protein kinase, purified Ab-MuLV kinase made in Escherichia coli was microinjected into Xenopus oocytes and was observed to cause a 7- to 15-fold increase in the phosphorylation of S6 on serine residues. Two-dimensional phosphopeptide maps of S6 phosphorylated in Ab-MuLV-transformed NIH cells in the absence of serum were identical to those of S6 isolated from normal cells grown in the presence of serum. In addition, S6 from oocytes injected with Ab-MuLV kinase yielded an S6 phosphopeptide map indistinguishable from that of serum-stimulated NIH 3T3 cells, whereas S6 from control oocytes lacked several phosphopeptides. Ab-MuLV kinase did not phosphorylate S6 directly in vitro, and microinjection of a mutant Ab-MuLV protein lacking kinase activity had no effect. These results indicate that the Ab-MuLV kinase interacts with a cellular pathway to enhance S6 phosphorylation by directly or indirectly activating an S6 protein kinase and/or inactivating an S6 protein phosphatase.
Inhibition of human platelet phospholipase A2 activity by unsaturated fatty acids.
Authors: Ballou L R , Cheung W Y
Published: May 2006

Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets increases significantly when the enzyme is separated from an endogenous inhibitor(s). The inhibitor, associated mainly with a particulate fraction, has now been identified as a mixture of unsaturated fatty acids. Treatment of the inhibitor with trypsin, RNase, DNase, or heat did not diminish its inhibitory activity, which was extractable by organic solvents. Incubation of PLA2 with phospholipids or various neutral lipids, including saturated fatty acids, had little or no effect on enzymatic activity. In contrast, unsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), all of which were detected in the particulate fraction, or longer chained unsaturated fatty acids inhibited PLA2 activity by 50% at approximately equal to 5 X 10(-7) M. The level of unsaturated fatty acids in the inhibitor fraction was equivalent to approximately equal to 10(-4) M, apparently sufficient to effectively inhibit PLA2 activity. Methylation of unsaturated fatty acids caused a complete loss of inhibitory activity, and subsequent demethylation restored the activity, suggesting that a free carboxyl group was necessary. Inhibition of PLA2 by unsaturated fatty acids appeared to be noncompetitive. PLA2 absolutely required Ca2+ for activity; the inhibition by unsaturated fatty acids was not reversed by Ca2+. The finding that unsaturated fatty acids are potent inhibitors of PLA2 would explain its generally low activity in human platelet extracts and its marked increase of activity during the course of enzyme purification.
Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells.
Authors: Falcone G , Tatò F , Alemà S
Published: May 2006

The relationship between susceptibility to transformation in vitro by different oncogenes and terminal differentiation was analyzed in embryonic quail myogenic cells. Infection with Rous sarcoma virus (RSV), Fujinami sarcoma virus (FSV), avian erythroblastosis virus (AEV), and the avian myelocytomatosis virus MC29 led to rapid and massive transformation. Transformed cells had distinctive morphological alterations, increased proliferation rates, and the ability to grow in agar suspension. Furthermore, homogeneously transformed cultures failed to fuse into multinucleated myotubes and to express muscle-specific genes. However, cloned populations of RSV-, FSV-, and AEV-transformed myogenic cells could, under appropriate culture conditions, partially differentiate into atypical "revertant" myotubes. In contrast, competence for terminal differentiation was completely and irreversibly suppressed on transformation by MC29. The specificity of action of a given oncogenic sequence on the inhibition of differentiation was further studied by using conditional and nonconditional transformation mutants. Myogenic cells infected with temperature-sensitive (ts) mutants of RSV and FSV exhibited a fully reversible block of differentiation after shift to restrictive temperature, while cells infected with ts34 AEV were not temperature sensitive for differentiation. Cultures infected with the partially transformation-defective mutant of MC29 td10H were morphologically transformed and acquired anchorage independence for proliferation but maintained a residual competence for terminal differentiation.
Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences.
Authors: Falkenthal S , Parker V P , Davidson N
Published: May 2006

The total sequence of the Drosophila melanogaster gene encoding the myosin light chain dissociated by alkali (MLC-ALK) has been determined. By sequence comparisons with an MLC-ALK cDNA clone and by S1 nuclease analyses, the pattern of introns and exons within the gene has been deduced. There are multiple polyadenylylation signals that can account for most of the observed heterogeneity in the lengths of mRNAs. In the 3' half of the gene, there are two alternative splicing patterns which result in mRNAs that translate to give proteins with two alternative 14 amino acid carboxyl-terminal sequences. There is developmental regulation of the selection of the above splicing sites. One splicing pattern produces an mRNA that translates into a protein used for both larval and adult musculature, whereas the other splicing pattern is used for the latter stage only.
Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid.
Authors: Downs S M , Coleman D L , Ward-Bailey P F , Eppig J J
Published: May 2006

Studies were carried out to identify and quantify the porcine follicular fluid (PFF) component(s) responsible for inhibition of murine oocyte maturation. A low molecular weight fraction of PFF (less than 1000) was prepared by dialysis and used in all experiments. This PFF fraction contained an inhibitor(s) of mouse oocyte maturation that absorbed maximally in the ultraviolet (UV) range at 250-260 nm. When the PFF fraction was charcoal-extracted, significant loss of absorbance at 250, 260, and 280 nm resulted, which corresponded to loss of inhibitory activity. Four major components of PFF were separated by ion-exchange chromatography and characterized according to their UV spectral characteristics and inhibitory activity. When individual fractions demonstrating identical spectra were pooled and analyzed by high-performance liquid chromatography, the first pooled fraction (A) was found to be impure, but adenine comprised 80% of the UV-absorbing material. Fractions B, C, and D were characterized as pure uracil, hypoxanthine, and 7-methylinosine, respectively. The concentrations of these compounds in PFF were estimated to be 0.06 mM adenine, 0.44 mM uracil, 1.41 mM hypoxanthine, and 0.19 mM 7-methylinosine. Comparison of the potencies of commercial preparations of these compounds established that hypoxanthine is the major inhibitory component of the low molecular weight PFF fraction. Moreover, a commercial preparation of hypoxanthine mimicked the action of PFF on mouse oocyte maturation in that it produced a transient inhibition of oocyte maturation that was significantly potentiated by follicle-stimulating hormone and dibutyryl cyclic adenosine monophosphate. When the inhibitory efficacies of purine and pyrimidine bases and nucleosides were compared, their relative potencies in decreasing order were purine bases greater than purine nucleosides greater than pyrimidine bases = pyrimidine nucleosides. We conclude that hypoxanthine is the predominant low molecular weight component of PFF that inhibits mouse oocyte maturation but that other purines/pyrimidines may also play a role in vivo in maintaining meiotic arrest.
A synthetic HIS4 regulatory element confers general amino acid control on the cytochrome c gene (CYC1) of yeast.
Authors: Hinnebusch A G , Lucchini G , Fink G R
Published: May 2006

Hybrid promoters constructed from upstream sequences of the yeast HIS4 promoter and the downstream element of the yeast CYC1 promoter place iso-1-cytochrome c (CYC1) expression under the general amino acid control, typical of HIS4. HIS4 fragments that confer regulation contain at least one copy of the sequence T-G-A-C-T-C that is repeated at HIS4 and other genes subject to the general control. A 14-base-pair synthetic oligonucleotide containing a single copy of the HIS4 repeat places CYC1 under the general control. Two copies of this oligonucleotide produce a derepressed level of expression nearly equivalent to that conferred by the largest HIS4 5' noncoding fragments we examined and direct regulated expression of a set of transcripts with 5' ends typical of the CYC1 promoter. Comparison of the expression levels conferred by the short synthetic repeat and larger HIS4 5' fragments reveals additional promoter elements required for maintaining efficient gene expression under repressing growth conditions.
Repair of defined single base-pair mismatches in
Escherichia coli
Authors: Dohet Christiane , Wagner Robert , Radman Miroslav
Published: May 2006

Heteroduplexes with single base-pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage λ DNA and used to transfect
Escherichia coli
. Each of the eight possible single base-pair mismatches was constructed. Genetic analysis of the progeny phages obtained from transfected bacteria indicates that the
E. coli
mismatch repair system does not recognize (or does not repair) all single base-pair mismatches with equal efficiency. In particular, the A·G, C·T, and C·C mismatches appear to be less repaired than any others. The mutator character of mismatch repair-deficient mutants suggests that such unrepaired mismatches should occur infrequently during
E. coli
DNA replication.
B cells that simultaneously express surface IgM and IgE in Nippostrongylus brasiliensis-infected SJA/9 mice do not provide evidence for isotype switching without gene deletion.
Authors: Katona I M , Urban J F , Finkelman F D
Published: May 2006

Recently, it has been reported that in SJA/9 mice infected with Nippostrongylus brasiliensis there are increased numbers of lymphoid cells positive for surface IgM and IgE (sIgM+ and sIgE+) even though they fail to secrete IgE, that both the sIgM and sIgE on these cells are intrinsic, and that there has been no deletion of genes for the Ig heavy chain constant region in these cells. These observations support a nondeletional model for Ig isotype switching. We have now reexamined the nature of sIgE on sIgE+ spleen and mesenteric lymph node cells of N. brasiliensis-infected SJA/9 mice, and the following observations lead us to believe that this sIgE is cytophilic rather than intrinsic: (i) Only approximately 50% of the N. brasiliensis-infected SJA/9 mice have detectable percentages of sIgE+ lymphoid cells. All mice with detectable sIgE+ lymphocytes have lymphocytes positive for intracytoplasmic IgE (cIgE+) and secrete IgE in vitro, while cIgE+ cells and IgE secretion are absent from N. brasiliensis-infected SJA/9 mice that lack sIgE+ cells. (ii) SJA/9 B lymphocytes have receptors for IgE: expression of these receptors is increased in N. brasiliensis-infected mice that have sIgE+ lymphocytes, but not in infected SJA/9 mice that lack sIgE+ lymphocytes. (iii) Treatment of sIgM+ sIgD+ sIgE+ cells for 1 min with dilute acid removes most sIgE but does not affect expression of sIgM or sIgD. (iv) The removal of mouse IgE from sIgE+ B cells facilitates the binding of exogenous rat IgE. (v) The small amount of sIgE that is reexpressed during a period of in vitro culture after acid treatment is blocked by inclusion of exogenous rat IgE in the culture medium. These observations show that most sIgM+ sIgE+ B cells in N. brasiliensis-infected SJA/9 mice do not express intrinsic sIgE; thus studies using these cells to determine mechanisms of Ig isotype switching are inconclusive.
Ascorbic acid mediates acetylcholine receptor increase induced by brain extract on myogenic cells.
Authors: Knaack D , Podleski T
Published: May 2006

Extracts of fetal calf brain cause a 3- to 5-fold increase in acetylcholine receptors (AcChoR) on cultured myogenic L5 cells. Purification of the substance causing the major portion of this receptor increase has been completed. Ultraviolet spectral characteristics, nuclear magnetic resonance, mass spectra, and AcChoR induction by the active factor are the same as those of commercially available ascorbic acid. The biological activity of ascorbic acid is not mimicked by reducing agents with or without sulfhydryl groups. Compounds related to ascorbic acid were tested for their ability to induce AcChoR increases on L5 cells. D-Isoascorbic acid is the only substance with identical biological activity to ascorbic acid. Dehydroascorbic acid and ascorbic acid 2-O-sulfate also induce AcChoR increases but with lower specific activity. These data show that ascorbic acid can play a role in regulating AcChoR expression in myogenic tissue, and the presence of ascorbic acid in the purified fraction from fetal calf brain accounts for its ability to increase AcChoR in L5 cells.
Choline acetyltransferase and acetylcholine in Xenopus oocytes injected with mRNA from the electric lobe of Torpedo.
Authors: Gundersen C B , Jenden D J , Miledi R
Published: May 2006

Xenopus oocytes were injected with poly(A)+ mRNA obtained from the electric lobes of Torpedo marmorata and Torpedo ocellata, which contain the cell bodies of the neurons that innervate the electric organs. The electric lobe mRNA preparation induces the oocytes to synthesize a catalytically active form of the enzyme choline acetyltransferase (EC 2.3.1.6). Enzymatic activity is found almost exclusively in the cytoplasmic fraction of injected, but not control, oocytes. Evidence is presented that distinguishes between the induced choline acetyltransferase activity and an intrinsic carnitine acetyltransferase activity present in the oocytes. This latter enzyme is associated principally with particulate fractions of the oocyte. The level of acetylcholine, which accumulates in mRNA-injected oocytes, is relatively insensitive to pharmacological manipulations that alter the acetylcholine content of other cells. These results show that Xenopus oocytes may be used advantageously to study functional properties of polypeptides associated with presynaptic elements in the nervous system.
Identification of the catalytic subunit of brain adenylate cyclase: a calmodulin binding protein of 135 kDa.
Authors: Coussen F , Haiech J , d'Alayer J , Monneron A
Published: May 2006

The partial purification of the eukaryote adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] catalytic subunit has been achieved by a procedure based on the calmodulin (CaM) sensitivity of the enzyme. Small amounts of rat brain synaptosomal membranes depleted of CaM were solubilized with Lubrol and subjected to a three-step chromatographic procedure involving gel filtration, a CaM-Sepharose affinity step, and fast protein liquid chromatography. About 20% of the adenylate cyclase activity contained in the membranes was recovered in the final enriched fraction with a specific activity of 200 nmol X mg-1 X min-1. The alpha subunits of the adenylate cyclase stimulatory proteins NS were absent from this final fraction. The addition of CaM, of forskolin, or of preactivated NS-containing fractions to this preparation greatly increased the enzyme activity. A CaM-binding polypeptide of 135,000 Da copurified with the adenylate cyclase activity in each of the three steps. Polyacrylamide gel electrophoresis of the final fraction showed that this polypeptide represented 35% of the total protein. We propose that this polypeptide is likely to be the adenylate cyclase catalytic subunit. This enzyme would represent close to 0.5% of the synaptosomal membrane proteins. Its low turnover number would be due to the absence of the alpha subunits of the NS regulatory proteins and would correspond to the enzymic basal level.
Evidence that an iron-nickel-carbon complex is formed by reaction of CO with the CO dehydrogenase from Clostridium thermoaceticum.
Authors: Ragsdale S W , Wood H G , Antholine W E
Published: May 2006

The interaction between carbon monoxide and the CO dehydrogenase from Clostridium thermoaceticum was studied by electron spin resonance (ESR) techniques. When the enzyme reacts with CO, a paramagnetic complex is formed which previously was shown, by isotope substitution, to be due to a nickel-carbon species. In this paper, we demonstrate that iron is also a component of this ESR-detectable complex. When the iron in the enzyme is replaced with 57Fe, a broadening of 18 G in the g parallel and 7 G in the g perpendicular region is seen. This hyperfine interaction is probably due to more than one iron atom in the complex. Coenzyme A influences this ESR spectrum. In the absence of CoA, the ESR spectrum consists of two superimposed signals, which were simulated using the following ESR parameters: signal 1, with g = 2.074 and g = 2.028, and signal 2 with gx = 2.062, gy = 2.047, and gz = 2.028. CoA converts signal 2 into signal 1. Since iron, nickel, and carbon all are part of this ESR-detectable complex, we propose that these atoms exist in a spin-coupled complex with net spin = 1/2, analogous to other iron-sulfur centers in which the metals are bridged by acid-labile sulfide.
Shape change of sickled erythrocytes induced by pulsed rf electrical fields.
Authors: Takashima S , Chang S , Asakura T
Published: May 2006

The shape change of sickled erythrocytes induced by electrical pulses was investigated under various experimental conditions using different suspending media. The purpose of this research is to find a set of conditions under which sickled erythrocytes from an individual with sickle cell anemia (SS erythrocytes) can be desickled with minimal damage to the membrane. Previously we observed, using 0.9% NaCl solution, that rounding of the SS erythrocytes eventually led to hemolysis even when the field was turned off. Using short pulses, we have now observed that the rounding of sickled erythrocytes does not necessarily lead to hemolysis if buffer solutions such as Hanks' solution are used. The cause of the shape change is believed to be the perforation of the erythrocyte membrane by the potential induced by applied fields. The field-induced membrane potential has been calculated for spherical cells and infinitely long cylinders. Using an ellipsoid of revolution as a model, we generalize the calculation of membrane potential. These calculations indicate that a transverse field is less effective in inducing a potential than a longitudinal field.
Gene amplification: an example of accelerated evolution in tumorigenic cells.
Authors: Sager R , Gadi I K , Stephens L , Grabowy C T
Published: May 2006

During selection for methotrexate resistance, tumorigenic CHEF/16 cells and derivatives from CHEF/16 tumors underwent amplification of the dihydrofolate reductase gene (DHFR) at accelerated rates compared with closely related nontumorigenic CHEF/18 cells. "Dot blot" analysis showed that the CHEF/16 cells contained many more copies of the DHFR gene than did the CHEF/18 cells, when assayed at similar elevated levels of methotrexate resistance. Chromosome analysis of cell samples taken at several time points during amplification revealed large differences between the nontumorigenic CHEF/18 cells and the two tumorigenic cell lines. The tumorigenic cells developed few chromosome rearrangements over a 4-log increase in methotrexate resistance, other than increased length of a single chromosome, which was shown by in situ hybridization to contain most or all of the amplified DHFR gene copies. In contrast, the CHEF/18 cells underwent complex, progressive changes in almost every chromosome, and in situ hybridization suggested a dispersed pattern of gene amplification. The data support the hypothesis that unregulated amplification is a pathological process, occurring readily in neoplastic but rarely in normal cells, that together with other chromosomal disturbances contributes to the rapid evolution and progression of cancer.
Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow.
Authors: Gendelman H E , Narayan O , Molineaux S , Clements J E , Ghotbi Z
Published: May 2006

Lentiviruses, as exemplified by visna virus of sheep, are nononcogenic retroviruses that cause slowly progressive diseases after prolonged periods of incubation. Earlier studies on visna have shown that the long incubation period of the disease is associated with constant production of minimal quantities of virus in tissues, whereas virus could be obtained by culturing monocytes and macrophages from explants of lymphatic tissues and inflamed organs. In this study the role of macrophages in lentivirus infection was explored using two sheep that were intrabronchially inoculated with virus. When sections of paraffin-embedded tissue, processed by a recently described technique which combines immunocytochemistry for the identification of macrophages and in situ hybridization for identification of viral nucleic acid, were examined, we found that virus replication is associated almost exclusively with infection in selected populations of macrophages in the interalveolar region of the alveoli, in inflammatory exudate cells in the lung, in lymph nodes, and in the spleen. Although large numbers of alveolar macrophages had viral RNA, few of these cells produced virus. While this minimally productive type of viral replication provides an explanation for the slow pace of the infection, restricted replication in terminally differentiated, short-lived macrophages does not explain persistent virus replication in the animal. With the discovery of clusters of infected macrophage precursors in the bone marrow, a mechanism for persistence was found. The macrophage precursor cells provide an important missing link in the virus-target-cell circuit and may be the reservoir of latently infected cells which perpetuate lentivirus infections in both animals and humans.
Modes of action of aspirin-like drugs.
Authors: Abramson S , Korchak H , Ludewig R , Edelson H , Haines K , Levin R I , Herman R , Rider L , Kimmel S , Weissmann G
Published: May 2006

Current dogma holds that nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibition of the synthesis and release of prostaglandins. However, NSAIDs also inhibit the activation of neutrophils, which provoke inflammation by releasing products other than prostaglandins. We now report that NSAIDs (e.g., indomethacin, piroxicam) inhibit activation of neutrophils by inflammatory stimuli, such as C5-derived peptides and leukotriene B4, even when cyclooxygenase products generated in suspensions of stimulated neutrophils (prostaglandin E and thromboxanes) are present. Sodium salicylate (3 mM) greatly inhibited aggregation of neutrophils but had no effect on aggregation of platelets or production of thromboxane induced by arachidonate. Sodium salicylate and other NSAIDs also inhibit calcium movements (45Ca uptake, changes in fluorescence of chlortetracycline and quin-2). Aspirin, sodium salicylate, indomethacin, and piroxicam also enhanced the poststimulation increase in intracellular cyclic AMP. NSAIDs therefore inhibit early steps in neutrophil activation as reflected by their capacity to inhibit movements of Ca and to enhance intracellular levels of cyclic AMP.
Mechanism of insulin action on membrane protein recycling: a selective decrease in the phosphorylation state of insulin-like growth factor II receptors in the cell surface membrane.
Authors: Corvera S , Czech M P
Published: May 2006

Insulin action in adipocytes leads to an increase in the steady-state number of cell surface glucose transporters and insulin-like growth factor II (IGF-II) receptors that appear to cycle continuously between the plasma membrane and a low-density membrane fraction. The IGF-II receptor could be labeled to constant specific activity by incubating adipocytes with [32P]phosphate for 2 hr. The extent of phosphorylation of IGF-II receptors in plasma membranes and in low-density microsomes was compared using 125I-labeled IGF-II binding and immunoblotting to quantitate the receptors present in each fraction. Receptors in the plasma membrane fraction of control cells incorporated approximately 1 molecule of phosphate per IGF-II binding site or 2 to 3 times more phosphate than was incorporated into IGF-II receptors in the low-density microsomes. Addition of insulin to labeled adipocytes did not change the specific activity of the gamma-phosphate of ATP but produced a specific and sharp decrease in the 32P-phosphate content of IGF-II receptors in the plasma membrane. No change due to insulin in the phosphorylation of receptors derived from low-density microsomes was observed. The insulin-mediated decrease in the [32P]phosphate content of IGF-II receptors from the plasma membrane was rapid in onset, paralleled the increase in the number of IGF-II receptors on the cell surface, and persisted for at least 30 min in the presence of insulin. Furthermore, when the effect of insulin to increase the number of IGF-II receptors in the cell surface was prevented by cooling cells to 5 degrees C, the decrease in phosphorylation of plasma membrane receptors could still be observed, indicating that this latter effect is not secondary to receptor redistribution. These data indicate that insulin inhibits one or more IGF-II receptor kinases or increases phosphatase activity, or both. Decreased phosphorylation of such insulin-sensitive plasma membrane components as IGF-II receptors may play a role in increasing their steady-state cell surface concentration, perhaps by delaying their internalization.
Cloning of Ly-5 cDNA.
Authors: Shen F W , Saga Y , Litman G , Freeman G , Tung J S , Cantor H , Boyse E A
Published: May 2006

A notable feature of Ly-5, among immunogenetic systems that identify glycoproteins of the cell surface and define the surface phenotype of cells according to their lineage, is that the Ly-5 locus specifies a range of molecular isoforms that distinguish cells of different stages and branches of hematopoietic development. The composition of the Ly-5 locus is of much interest in regard to how these isoforms are constructed and differentially regulated according to cell lineage. We describe here a cDNA clone, pLy-5-68, that identifies Ly-5. The Ly-5 specificity of the pLy-5-68 clone was first indicated by a restriction fragment length polymorphism (RFLP), which in Southern blotting distinguishes genomic DNA of C57BL/6 (B6) mice (Ly-5a) from that of B6-Ly-5b congeneic mice whose genome is the same as B6 except for the segment of chromosome 1 that bears Ly-5b. For the following reasons it is unlikely that pLy-5-68 represents a gene linked to Ly-5 that was carried over with Ly-5b during serial backcrossing to make the B6-Ly-5b congeneic strain. In all mouse strains tested, the serological Ly-5 allotype (Ly-5.1 vs. Ly-5.2) accorded with the RFLP pattern. Cells of the ST/bJ mouse strain have unique Ly-5 serological reactions and ST/bJ DNA gives a unique (third) RFLP pattern (Ly-5c) with pLy-5-68. All Ly-5+ cell types reacted positively with pLy-5-68 in RNA transfer blotting, and all Ly-5- cell types tested did not. The difference in size of mRNA reactive with pLy-5-68 in cells expressing the 200-kDa Ly-5 isoform as compared with cells expressing the 220-kDa Ly-5 isoform corresponded with the difference in size of the protein components of those isoforms.
Suppression of retroviral propagation and disease by suramin in murine systems.
Authors: Ruprecht R M , Rossoni L D , Haseltine W A , Broder S
Published: May 2006

Retroviral propagation crucially depends on reverse transcriptase (RT). We have developed murine models to test the biological effectiveness of the RT inhibitor suramin. The drug was active in our assay system, which includes (i) inhibition of RT activity in the murine T-cell tropic virus SL3-3 and Rauscher murine leukemia virus (MuLV), (ii) inhibition of plaque formation in the XC plaque assay, (iii) inhibition of viral infection of cultured murine T cells, and (iv) inhibition of splenomegaly induced by Rauscher MuLV in BALB/c mice. Suramin decreases viral titers significantly, even if started 36 hr after infection. Viral titers and number of infected cells increased to control levels after removal of the drug. BALB/c mice treated i.v. with 40 mg of suramin per kg twice per week following infection with Rauscher MuLV showed a 35% decrease in splenomegaly. Suramin is an active antiretroviral agent whose effect on retroviral propagation is reversible. We conclude that it acts as a virustatic drug and that long-term administration of suramin will be necessary if it is used for experimental treatment of human retroviral illnesses such as the acquired immune deficiency syndrome.
Single anion-selective channels in basolateral membrane of a mammalian tight epithelium.
Authors: Hanrahan J W , Alles W P , Lewis S A
Published: May 2006

Basolateral membrane chloride permeability of surface cells from rabbit urinary bladder epithelium was studied using the patch-clamp technique. Two types of anion-selective channel were observed. One channel type showed inward rectification and had a conductance of 64 pS at-50 mV when bathed symmetrically by saline solution containing 150 mM chloride; the other resembled high-conductance voltage-dependent anion channels (VDACs). Both channels had the selectivity sequence Cl-approximately equal to Br-approximately equal to I- approximately equal to SCN- approximately equal to NO3- greater than F- greater than acetate greater than gluconate greater than Na+ approximately equal to K+ and were sensitive to the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Basolateral chloride conductance in urinary bladder is apparently due to the 64 pS anion channel, which is active at physiological potentials. Imperfect selectivity of this channel against cations might also account for the low, but finite, sodium permeability of the basolateral membrane.
Ionic currents that generate the spontaneous diastolic depolarization in individual cardiac pacemaker cells.
Authors: Shibata E F , Giles W R
Published: May 2006

An enzymatic dispersion procedure has been developed to obtain viable, spontaneously active single myocytes from cardiac pacemaker tissue: the bullfrog (Rana catesbeiana) sinus venosus. Recordings of time- and voltage-dependent Ca2+ and K+ currents have been made by using a single suction-microelectrode technique. The results show that two time- and voltage-dependent currents interact to modulate the slope of the pacemaker potential. These are: (i) the decay of a delayed rectifier K+ current and (ii) the activation of a Ca2+ current. In addition, the data strongly suggest that cardiac pacemaker tissue does not have an inwardly rectifying background K+ current.
Expression of murine 21-hydroxylase in mouse adrenal glands and in transfected Y1 adrenocortical tumor cells.
Authors: Parker K L , Chaplin D D , Wong M , Seidman J G , Smith J A , Schimmer B P
Published: May 2006

The S region of the murine major histocompatibility complex contains two structurally related genes (21-OHase A and 21-OHase B) that encode 21-hydroxylase (21-OHase), an enzyme essential for the synthesis of adrenal steroids. Expression of these two genes has been analyzed by using oligonucleotide probes specific for the 21-OHase A and B genes and by DNA-mediated gene transfer. Hybridization of the oligonucleotides to blots of BALB/c adrenal RNA demonstrated that all 21-OHase mRNA is derived from the 21-OHase A gene. Cosmids bearing either the 21-OHase A or B gene were introduced into Y1 adrenocortical tumor cells by cotransfection with pSV2-neo. Cells transfected with the 21-OHase A gene expressed 21-OHase as determined by steroid metabolism and by RNA blot hybridization; 21-OHase transcripts were not detected in parent Y1 cells or in cells transfected with the 21-OHase B gene. Treatment of 21-OHase A transfectants with adrenocorticotropin increased 21-OHase mRNA levels by up to 10-fold, thus mimicking the observed effect of this hormone on 21-OHase levels in primary adrenal cultures. The regulated expression of the 21-OHase A gene in transfected Y1 cells should provide a useful system for the investigation of factors controlling the adrenal-specific regulation of 21-OHase activity.
Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I.
Authors: Bateman E , Iida C T , Kownin P , Paule M R
Published: May 2006

The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from the transcription start site, protecting a region extending from around -14 to -67 on the coding strand, and -12 to -69 on the noncoding strand. The protein that binds to DNA within this region can be competed out by using wild-type promoters but not by using mutants which do not stably bind the factor. RNA polymerase I can form a stable complex in the presence of DNA and transcription factor, allowing footprinting of the complete transcription initiation complex. RNA polymerase I extends the protected region obtained with TIF alone to around +18 on the coding strand, and to +20 on the noncoding strand. This region is not protected by polymerase I in the absence of TIF. The close apposition of the regions protected by TIF and polymerase provides evidence that accurate transcription of the ribosomal gene may be achieved through protein-protein contacts as well as through DNA-protein interactions.
Activation of Na+/H+ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca2+ ionophore, and growth factors.
Authors: Vicentini L M , Villereal M L
Published: May 2006

The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H+ exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H+ exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts.
Cortical anomalies in brains of New Zealand mice: a neuropathologic model of dyslexia?
Authors: Sherman G F , Galaburda A M , Geschwind N
Published: May 2006

Cortical anomalies have been reported in the brains of dyslexic individuals. In addition, dyslexic and left-handed individuals have a higher than expected rate of some immune-related diseases. The possible association between immune and cerebrocortical pathology was investigated in the immune-defective New Zealand Black mouse and its hybrid with the New Zealand White mouse. Structural anomalies similar to those present in the dyslexic brain were seen in the brains of these mice.
Structure, evolution, and regulation of a fast skeletal muscle troponin I gene.
Authors: Baldwin A S , Kittler E L , Emerson C P
Published: May 2006

The complete structure of a quail fast skeletal muscle troponin I gene was determined by nucleotide sequence comparison of troponin I genomic and cDNA sequences. This 4.5-kilobase troponin I gene has eight exons. The actin-binding domain of troponin I is encoded by a single exon, whereas the troponin C-binding domain is split into at least two exons. The exon organization of the fast troponin I gene suggests that gene conversion directs the nonrandom conservation of the carboxyl-terminal halves of troponin I isoforms and that the amino-terminal extension of the cardiac isoform originated by splice-junction sliding. Comparison of the structure of the troponin I gene with the structures of other contractile protein genes reveals homologous sequences in their 5' flanking regions and similar large introns that separate protein-coding exons from 5' nontranslated exons. These common structural features may function to coordinate the activation of contractile-protein genes during myogenesis.
Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T lymphocytes.
Authors: Cantrell D A , Davies A A , Crumpton M J
Published: May 2006

As judged by indirect immunofluorescence, phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol induced a rapid, concentration-dependent decrease of about 50% in the surface expression of the T3 antigen on human T lymphoblasts, and of T3 and the T-cell antigen receptor on HPB-ALL cells. Direct binding experiments using 125I-labeled antibody indicated that the reduction in T3 expression corresponded to a decrease in the number of antigen molecules rather than a change in their affinity. Biochemical analyses revealed that phorbol dibutyrate induced a rapid, prominent phosphorylation of the T3 Mr 26,000 gamma chain and to a lesser extent of the Mr 21,000 delta chain. No phosphorylation of the T3 epsilon chain or of the alpha and beta subunits of the T-cell antigen receptor was detected. The data suggest that protein kinase C induces a phosphorylation of the T3 gamma and delta chains that may lead to the down-regulation of the T3/T-cell antigen receptor complex.
Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.
Authors: Smith G E , Ju G , Ericson B L , Moschera J , Lahm H W , Chizzonite R , Summers M D
Published: May 2006

A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.
Upstream promoter element of the human metallothionein-IIA gene can act like an enhancer element.
Authors: Haslinger A , Karin M
Published: May 2006

Initiation of transcription by RNA polymerase II in eukaryotes is strongly increased by cis-acting genetic elements, known as activators or enhancers. Enhancers, first detected in simian virus 40 (SV40), were subsequently also found to control the expression of several cellular genes. The human metallothionein-IIA (hMT-IIA) gene, although inducible by heavy metals and glucocorticoids, is widely expressed in most cell types in the absence of inducers. Here we show that the high basal level of transcription of the hMT-IIA gene is due to the presence of an enhancer element within the hMT-IIA promoter region. The structural and functional organization of this cellular enhancer element in two direct repeats is strikingly similar to that of the enhancer element of SV40. This suggests a possible functional and evolutionary relationship between enhancers and upstream promoter elements.
Sequence-directed mutagenesis: evidence from a phylogenetic history of human alpha-interferon genes.
Authors: Golding G B , Glickman B W
Published: May 2006

We have studied the potential contribution of template-dependent events to genetic variation in mammals by examining the sequence alterations that have occurred in the recent evolution of human interferon genes. Fifteen members of the human alpha-interferon gene family were aligned, and a phylogenetic history was inferred. Many multiple events are inferred to have occurred in the evolution of the interferon genes and for the majority of these local DNA sequences were present that were capable of serving as templates for their occurrence. We conclude that the DNA sequence has the potential to explain many of the inferred spontaneous events and to explain complex alterations to sequences--i.e., the joint occurrence of base substitutions and insertions/deletions. Thus, such a mechanism would often cause multiple sequence changes as a result of a single mutational event and would provide additional genetic variation for evolution. Sequence-directed mutations would depend upon the local DNA sequences and, hence, would not be random at the DNA level.