bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoregulation of rhodopsin synthesis in Chlamydomonas reinhardtii.
Authors: Foster K W , Saranak J , Zarrilli G
Published: May 2006

A sensitive assay for the induction of carotenoid and rhodopsin synthesis, based on the phototactic response, has been developed in a mutant of the unicellular alga Chlamydomonas reinhardtii. In the dark, the mutant fails to synthesize carotene and retinal, but it contains the apoprotein opsin. When retinal synthesis is induced by light treatment, the retinal combines with opsin to form rhodopsin, and the cells swim away from a source of light. Since the amount of light required to trigger a phototactic response is inversely proportional to the concentration of rhodopsin, the decrease in amount of light necessary to generate that response can serve as a measure of the amount of retinal synthesized in cells after induction. Using this assay, we found that (i) light induction of retinal depends linearly on light exposure and rhodopsin concentration during the exposure; (ii) the action spectrum of light induction is identical with that for phototaxis for which the receptor pigment is rhodopsin; and (iii) incubation with all-trans-7,8-dihydroretinal before light exposure shifts the action-spectrum peak for light induction 0.41 eV (-71 nm). We conclude that the photopigment for induction of retinal synthesis is a rhodopsin. The time lag required for induction of retinal synthesis and preliminary experiments with transcription or translation inhibitors suggest that alterations in gene expression could be involved in the induction process. Its control could be similar to other processes in which membrane receptors for hormones, neurotransmitters, or growth factors regulate gene expression.
Increased mRNA expression of a laminin-binding protein in human colon carcinoma: complete sequence of a full-length cDNA encoding the protein.
Authors: Yow H K , Wong J M , Chen H S , Lee C G , Davis S , Steele G D , Chen L B
Published: May 2006

Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers we constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here we report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is approximately equal to 9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. Containing only two cysteine residues, the protein does not have consensus sequences for asparagine-linked glycosylation, amphipathic alpha-helix, or the N-terminal leader signal sequences for entry into endoplasmic reticulum, although hydrophobic segments for potential membrane associations exist. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant (no lysine or arginine) and highly negatively charged (13 aspartic plus glutamic residues). Within this segment are five repeats of (Asp/Glu)-Trp-(Ser/Thr); two of these are nearly tandem repeats of Thr-Glu-Asp-Trp-Ser-Ala-Xaa-Pro.
A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.
Authors: Moyzis R K , Buckingham J M , Cram L S , Dani M , Deaven L L , Jones M D , Meyne J , Ratliff R L , Wu J R
Published: May 2006

A highly conserved repetitive DNA sequence, (TTAGGG)n, has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.
Participation of Escherichia coli integration host factor in the P1 plasmid partition system.
Authors: Funnell B E
Published: May 2006

Stable maintenance of the plasmid prophage of bacteriophage P1 requires the P1 ParB protein, which acts on a DNA site termed parS. Fractionation of extracts from Escherichia coli cells overproducing ParB revealed that a host factor, in addition to ParB, is required to observe maximal binding to parS, as detected by a nitrocellulose filter retention assay. Two observations indicated that this factor is E. coli integration host factor (IHF): purified IHF substituted specifically for host factor from a crude lysate, and lysates prepared from cells deficient in the beta subunit of IHF (E. coli hip mutants; also called himD) contained no host factor activity. Binding studies in vitro and competition experiments in vivo suggest that two types of ParB-parS DNA complexes can exist that differ in (i) the presence of IHF, (ii) the amount of parS sequence with which the proteins interact, and (iii) the specificity of their participation in partition. Under normal conditions, with the intact P1 partition region and wild-type bacteria, P1 plasmids apparently use IHF to assist ParB in the assembly of a functional partition complex at parS.
Measurement of outward translocation of phospholipids across human erythrocyte membrane.
Authors: Bitbol M , Devaux P F
Published: May 2006

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.
Activation of the fructose 1,6-bisphosphatase gene by 1,25-dihydroxyvitamin D3 during monocytic differentiation.
Authors: Solomon D H , Raynal M C , Tejwani G A , Cayre Y E
Published: May 2006

Cells from the human leukemia cell line HL-60 undergo terminal monocyte-like differentiation after exposure to either the active circulating form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or phorbol 12-myristate 13-acetate. Little is known about the genes that regulate monocytic differentiation. Using clonal variant cells of HL-60 origin, we constructed a cDNA library enriched for genes that are induced by 1,25-(OH)2D3. We now report that in HL-60, the fructose 1,6-bisphosphatase (FBPase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) gene is activated during 1,25-(OH)2D3-induced monocytic differentiation. This gene encodes two closely related mRNAs; one, activated by 1,25-(OH)2D3 at an early stage of HL-60 differentiation, encodes a protein that has homology to mammalian FBPase, a key enzyme in gluconeogenesis, although it does not exhibit its classical enzymatic activity. A second mRNA is activated by 1,25-(OH)2D3 mainly in peripheral blood monocytes. This mRNA is present in kidney as a unique transcript and encodes a protein with FBPase activity. Our data also show that this FBPase-encoding mRNA can be activated during monocytic maturation since it was detected in human alveolar macrophages.
Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.
Authors: Trapani J A , Klein J L , White P C , Dupont B
Published: May 2006

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity.
Molecular cloning of a cDNA coding biliary glycoprotein I: primary structure of a glycoprotein immunologically crossreactive with carcinoembryonic antigen.
Authors: Hinoda Y , Neumaier M , Hefta S A , Drzeniek Z , Wagener C , Shively L , Hefta L J , Shively J E , Paxton R J
Published: May 2006

We have isolated and sequenced four overlapping cDNA clones from a normal adult human colon library, which together gave the entire nucleotide sequence for biliary glycoprotein I (BGP I). BGP I is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily in the immunoglobulin gene superfamily. The deduced amino acid sequence of the combined clones for BGP I revealed a 34-residue leader sequence followed by a 108-residue N-terminal domain, a 178-residue immunoglobulin-like domain, a 108-residue region specific to BGP I, a 24-residue transmembrane domain, and a 35-residue cytoplasmic domain. The nucleotide sequence of BGP I exhibited greater than 80% identity with CEA and nonspecific crossreacting antigen (NCA) in the leader peptide, N-terminal domain, and immunoglobulin-like domain. The BGP I-specific domain, designated A', was 56.7% and 55.8% identical at the nucleotide level and 42.6% and 39.6% identical at the amino acid level to the immunoglobulin-like domain of NCA and the first immunoglobulin-like domain of CEA, respectively. Beyond nucleotide position 1375 the 3' region of the BGP I cDNA was found to be specific for BGP I. Hybridization of a probe from this region to electrophoretic blots of RNAs from different human tissues showed a predominant 2.8-kilobase (kb) message accompanied by weaker bands 4.1 and 2.1 kb in size. The same probe gave a single band in Southern blot analysis of restricted total human DNA. Using a coding region probe from the BGP I domain A', we observed 4.1- and 2.1-kb messages. Lack of the 2.8-kb band suggested that different forms of BGP I may be generated by posttranscriptional modification of the same gene. We propose that BGP I diverged from NCA by acquiring an immunoglobulin-like domain substantially different from the domains found in NCA or CEA and also a new cytoplasmic domain. The latter feature should result in a substantially different membrane anchorage mechanism of BGP I compared to CEA, which lacks the cytoplasmic domain and is anchored via a phosphatidylinositol-glycan structure. Protein structural analysis of BGP I isolated from human bile revealed a blocked N terminus, 129 amino acids of internal sequence that are in agreement with the translated cDNA sequence, and five glycosylation sites in the peptides sequenced.
Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells.
Authors: Chiu C P , Moulds C , Coffman R L , Rennick D , Lee F
Published: May 2006

Interleukin 6 (IL-6) refers to the gene product that was characterized initially as beta 2 interferon/26-kDa protein produced by human fibroblasts and later was found to be identical to B-cell stimulatory factor 2, hybridoma/plasmacytoma growth factor, and probably hepatocyte-stimulating factor. Using the human IL-6 cDNA as a probe, we have isolated functional cDNA clones from mouse bone marrow stromal cell cDNA libraries. Sequence analysis of the mouse cDNA insert revealed significant homology between the human and mouse IL-6 cDNA clones both at the level of nucleotide (65%) and deduced amino acid (41%) sequences. The NH2-terminal sequence of the deduced protein is identical to a partial NH2-terminal sequence determined previously for a hybridoma/plasmacytoma growth factor and a plasmacytoma growth factor isolated from mouse T cells and macrophages, respectively. The mRNA for mouse IL-6 is expressed in IL-1-treated stromal cells and in activated T-cell and macrophage cell lines. Supernatants from COS-7 monkey cells transfected with the cDNA clone have plasmacytoma growth factor, hepatocyte-stimulating factor, and colony-stimulating factor activities, as well as the ability to support the growth of a factor-dependent myeloid cell line, thus revealing an additional biological activity for IL-6.
Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes.
Authors: Srivastava A K , Schlessinger D
Published: May 2006

In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated.
Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors.
Authors: Chang C S , Kokontis J , Liao S T
Published: May 2006

Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural ARs. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.
Identification of a functional mammalian spliceosome containing unspliced pre-mRNA.
Authors: Abmayr S M , Reed R , Maniatis T
Published: May 2006

Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.
Growth regulation of cancer metastases by their host organ.
Authors: Sargent N S , Oestreicher M , Haidvogl H , Madnick H M , Burger M M
Published: May 2006

We analyzed mechanisms responsible for organ-specific metastasis by using two melanoma sublines derived from the same mouse tumor, of which one colonizes the lungs (F10) and the other colonizes the liver (L8) after intravenous injection. Both lines were obtained by selective growth in lung or liver after injection of tumor cells into a tail vein or portal vein. Contrary to common concepts, the cells of the liver-colonizing melanoma line do not accumulate preferentially in the liver after intravenous administration in vivo. However, the selective survival and proliferation of these melanoma cells in the target organ (liver) may be explained by the unexpected observation that they can be specifically stimulated to proliferate in the presence of hepatocytes, whereas the cells of the lung-colonizing line cannot. Growth promotion under coculture conditions in vitro was monitored both by thymidine incorporation into DNA and by increase in cell numbers. The proliferative stimulus is not mediated by an easily diffusible factor but rather depends upon direct contact between liver cells and those tumor cells that metastasize to that particular organ.
Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA.
Authors: Fox T D , Sanford J C , McMullin T W
Published: May 2006

The mitochondrial gene oxi1, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rho0). The plasmid DNA behaved in a manner entirely consistent with the known properties of normal yeast rho- mtDNA after its introduction by high-velocity microprojectile bombardment. Like the mtDNA sequences retained in natural rho- strains, the plasmid DNA in the transformants was reiterated into concatemers whose size was indistinguishable from that of wild-type mtDNA. The oxi1 sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxi1 genetic information in these "synthetic rho-" strains could be expressed in diploids either after "marker rescue" by recombination with rho+ mtDNA carrying an appropriate oxi1 point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxi1 sequences and rho+ mtDNA with oxi1 deleted. The ability to generate such "synthetic rho-" strains by transformation will allow transfer of mutations generated in vitro to wild-type rho+ mtDNA as well as examination of the function of altered genes in trans.
"DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.
Authors: Goldstein A , Nestor J J , Naidu A , Newman S R
Published: May 2006

We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.
Extinction dynamics of age-structured populations in a fluctuating environment.
Authors: Lande R , Orzack S H
Published: May 2006

We model density-independent growth of an age- (or stage-) structured population, assuming that mortality and reproductive rates fluctuate as stationary time series. Analytical formulas are derived for the distribution of time to extinction and the cumulative probability of extinction before a certain time, which are determined by the initial age distribution, and by the infinitesimal mean and variance, mu and sigma 2, of a diffusion approximation for the logarithm of total population size. These parameters can be estimated from the average life history and the pattern of environmental fluctuations in the vital rates. We also show that the distribution of time to extinction (conditional on the event) depends on the magnitude but not the sign of mu. When the environmental fluctuations in vital rates are small or moderate, the diffusion approximation gives accurate estimates of cumulative extinction probabilities obtained from computer simulations.
Cooperative DNA binding of the yeast transcriptional activator GAL4.
Authors: Giniger E , Ptashne M
Published: May 2006

The yeast transcriptional regulatory protein GAL4 binds to four sites in the GAL upstream activating sequence and stimulates transcription of the adjacent GAL1 and GAL10 genes. We show here that binding to at least two of these sites is cooperative in vivo. We also measure stimulation of transcription by pairs of GAL4 binding sites and find that the activities of low-affinity binding sites combine synergistically, whereas the activities of high-affinity binding sites combine only additively. We suggest that the synergistic transcriptional stimulatory activity of the naturally occurring GAL4 binding sites is solely a manifestation of cooperative binding of GAL4 protein to DNA and that the activity of a GAL upstream activating sequence is roughly proportional to the number of GAL4 molecules bound.
Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene
Authors: Cheung Alice Y. , Bogorad Lawrence , Van Montagu Marc , Schell Jeff
Published: May 2006

The chloroplast gene
psbA
codes for the photosynthetic quinone-binding membrane protein Q
B
, which is the target of the herbicide atrazine. This gene has been converted into a nuclear gene. The
psbA
gene from an atrazine-resistant biotype of
Amaranthus hybridus
has been modified by fusing its coding region to transcription-regulation and transit-peptide-encoding sequences of a bona fide nuclear gene. The constructs were introduced into the nuclear genome of tobacco by using the
Agrobacterium
tumor-inducing (Ti) plasmid system, and the protein product of nuclear
psbA
has been identified in the photosynthetic membranes of chloroplasts. Recovery of atrazine-tolerant transgenic plants shows that the product of the transplanted gene functions in photosynthesis. These experiments show that it is possible to modify chloroplast-gene-specified functions via nuclear-genome transformation and also raise evolutionary questions.
Cloning of the cDNA for human 5-lipoxygenase.
Authors: Dixon R A , Jones R E , Diehl R E , Bennett C D , Kargman S , Rouzer C A
Published: May 2006

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two reactions in the pathway leading to the formation of leukotrienes from arachidonic acid. Leukotrienes are potent arachidonic acid metabolites possessing biological activities that suggest a role in the pathophysiology of allergic and inflammatory diseases. To obtain structural information about 5-LO for use in developing anti-inflammatory chemotherapeutic agents, the enzyme was purified from human polymorphonuclear leukocytes and the amino acid sequences were determined for several cyanogen bromide-derived peptides. A cDNA clone encoding a 674-amino acid protein containing all of the derived peptide sequences was isolated from a dimethyl sulfoxide differentiated HL60 cell cDNA library. The mRNA encoding 5-LO was detected only in differentiated HL60 cells and not in the undifferentiated cell line, indicating that the expression of 5-LO in this cell line is transcriptionally regulated. In addition, the human protein displays some amino acid sequence homology with several lipases and significant homology with the partial sequences of soybean and reticulocyte lipoxygenases. Thus, 5-LO appears to be a member of a larger family of related enzymes.
Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatin core.
Authors: Baker T S , Drak J , Bina M
Published: May 2006

The three-dimensional structure of the capsid and the nucleohistone core of simian virus 40 (SV40) has been reconstructed by image analysis of electron micrographs of frozen hydrated samples. The 72 prominent capsomere units that comprise the T = 7d icosahedral surface lattice of the capsid are clearly resolved. Both the pentavalent and hexavalent capsomeres appear with pentameric substructure, indicating that bonding specificity in the shell is not quasi-equivalent. There is a remarkable similarity between the structure of the SV40 virion capsid and the structure reported for the polyoma empty capsid. This result establishes that (i) the unexpected pentameric substructure of the hexavalent capsomeres is also present in virions and (ii) the arrangement of the 72 pentamers in the capsid lattice may be a characteristic feature of the entire papova family of viruses. The center of the SV40 reconstruction reveals electron density corresponding to the nucleohistone core. This density is smeared, suggesting that the minichromosome is not organized with icosahedral symmetry matching the capsid symmetry. The visualization of the virion chromatin provides a basis for invoking new models for the higher order structure of the encapsidated minichromosome.
Closely linked early and late histone H2B genes are differentially expressed after microinjection into sea urchin zygotes.
Authors: Colin A M , Catlin T L , Kidson S H , Maxson R
Published: May 2006

An early and a late histone H2B gene from the sea urchin Stronglyocentrotus purpuratus were linked in a single plasmid and injected into the eggs of the sea urchin Lytechinus pictus. The levels of transcripts of injected early and late genes and of endogenous early genes were monitored during development by a ribonuclease protection assay. Transcripts of both the injected and endogenous early genes peaked during the blastula stage and decreased severalfold by the mesenchyme blastula stage. Transcripts of the injected late gene became detectable at the blastula stage and increased in amount subsequently, until at least the early gastrula stage, 28 hr after fertilization. Thus, the pattern of expression of the injected early and late H2B genes is similar to that of their endogenous counterparts. These results show that DNA sequences regulating the temporal pattern of early and late H2B gene expression must lie within the cloned DNA segments; i.e., within 600 base pairs of the early H2B gene and 3 kilobases of the late H2B gene.
Receptor-like function of heparin in the binding and uptake of neutral lipids.
Authors: Bosner M S , Gulick T , Riley D J , Spilburg C A , Lange L G
Published: May 2006

Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.
The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.
Authors: Bauer G A , Burgers P M
Published: May 2006

DNA polymerase III from Saccharomyces cerevisiae is analogous to the mammalian DNA polymerase delta by several criteria, including an increased synthetic activity on poly(dA).oligo(dT) (40:1 nucleotide ratio) in the presence of calf thymus proliferating-cell nuclear antigen (PCNA), or cyclin. This stimulation assay has been used to purify the yeast analog of PCNA/cyclin (yPCNA) to homogeneity. yPCNA is a trimer or tetramer (Mr approximately 82,000) of identical subunits with a denatured Mr of 26,000. On a molar basis yPCNA and calf thymus PCNA/cyclin are equally active in stimulating DNA synthesis by DNA polymerase III. About 10 times more yPCNA than calf thymus PCNA/cyclin is needed, however, to stimulate calf thymus DNA polymerase delta, and the degree of stimulation obtained at saturating levels of yPCNA is a factor of 2-3 less than with calf thymus PCNA/cyclin. Both stimulatory proteins exert their effect in an identical fashion, i.e., by increasing the processivity of the DNA polymerase. Yeast DNA polymerases I and II and calf thymus DNA polymerase alpha are not stimulated by yPCNA. Treatment of logarithmic-phase cells with hydroxyurea blocks them in the S phase and produces a 4- to 5-fold increase in yPCNA.
Dynamic properties of membrane proteins: reversible insertion into membrane vesicles of a colicin E1 channel-forming peptide.
Authors: Xu S , Cramer W A , Peterson A A , Hermodson M , Montecucco C
Published: May 2006

The binding of colicin E1 and its COOH-terminal channel-forming peptides to artificial membrane vesicles has an optimum at acidic pH values. The Mr 18,000 thermolytic peptide inserted into membrane vesicles at pH 4.0 has a limited accessibility to exogenous protease. It is converted by trypsin cleavage after Lys-381 and Lys-382 to a lower Mr 14,000 peptide. However, when the pH of a vesicle suspension to which peptide has been bound at pH 4.0 is shifted to 6.0, the accessibility to protease increased greatly. This was shown (i) by the large decrease in the amount of Mr 14,000 or Mr 18,000 peptide after the pH 4----6 shift and treatment with trypsin or Pronase, consistent with (ii) a previously observed decrease in membrane-bound radiolabeled peptide after protease treatment. (iii) When a photoactivable nitrene-generating phospholipid probe was used to label the colicin peptide inserted into the bilayer, the extent of labeling decreased by a factor of 3 when the pH was shifted from 4.0 to 6.5. (iv) Colicin peptide added to vesicles at pH 4.0 can "hop" to other vesicles if the pH and ionic strength of donor vesicles are successively increased. It is proposed that deprotonation of acidic residues in contact with the hydrophobic bilayer or the membrane surface destabilizes the inserted channel and causes it to be extruded from the membrane. The pH-dependent extrusion of the inserted colicin channel provides an example of dynamic properties of an intrinsic membrane protein.
A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction.
Authors: Corvera S , Folander K , Clairmont K B , Czech M P
Published: May 2006

Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of approximately 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at approximately 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane, as detected by immunoblotting with a monoclonal antibody. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated approximately equal to 3-fold in this clathrin-enriched subfraction. Insulin treatment of intact cells increased the total IGF-II/Man-6-P receptors in the Triton X-100-soluble fraction of the plasma membrane, whereas no change in receptor number in the detergent-insoluble fraction was seen. However, insulin markedly decreased the phosphorylation stoichiometry of the Triton X-100-insoluble receptors. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction.
Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.
Authors: Payne J , Huber B T , Cannon N A , Schneider R , Schilham M W , Acha-Orbea H , MacDonald H R , Hengartner H
Published: May 2006

Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA.
Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of
Solemya velum
(Bivalvia: Mollusca)
Authors: Cavanaugh Colleen M. , Abbott Marilyn S. , Veenhuis Marten
Published: May 2006

The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (Rbu
P
2
Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of
Solemya velum
, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of Rbu
P
2
Case from tobacco (
Nicotiana tabacum
) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of
S. velum
. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of
Thiobacillus neapolitanus
, a free-living sulfuroxidizing chemoautotroph whose Rbu
P
2
Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the Rbu
P
2
Case activity.
Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord.
Authors: De Biasi S , Rustioni A
Published: May 2006

By light microscopic immunocytochemistry it has been previously shown that approximately equal to 70% of the neurons in rat dorsal root ganglia are labeled with an antiserum for glutamate conjugated to hemocyanin; the smaller among these neurons are also positive for substance P. By using a postembedding ImmunoGold method and electron microscopy, it is shown here that synaptic terminals in the superficial laminae of the spinal cord of rats selectively stain for the same glutamate antiserum. Immunolabeling is in small dome-shaped and in large scalloped synaptic terminals. Scalloped terminals are of two types. One type consists of dark terminals with many agranular vesicles of different size and a few large granular vesicles; these are probably endings of unmyelinated and small myelinated primary afferent fibers. The other type consists of light terminals with small agranular vesicles homogeneous in size with neurofilaments and many mitochondria; these are probably endings of larger myelinated primary afferent fibers. By means of double-labeling electron microscopic immunocytochemistry with colloidal gold particles of two different sizes, it is also shown here that substance P is present in only the dark type of glutamate-labeled scalloped terminals. The primary afferent origin of the terminals labeled by the antisera for glutamate and for substance P is demonstrated by a triple-labeling strategy: immunocytochemistry for both antisera on sections from rats in which dorsal rhizotomy or dorsal root ganglion injection of horseradish peroxidase conjugated to wheat germ agglutinin was performed. It is proposed that glutamate is the neurotransmitter in primary afferents mediating input from different peripheral receptor classes, including nociceptors. Effects of glutamate and substance P on spinal dorsal horn neurons may result from co-release of these two mediators from the same dorsal root afferent terminal.
Expression of a bovine rhodopsin gene in Xenopus oocytes: demonstration of light-dependent ionic currents.
Authors: Khorana H G , Knox B E , Nasi E , Swanson R , Thompson D A
Published: May 2006

Xenopus oocytes express a gene encoding bovine rhodopsin as well as its SP6 RNA polymerase-derived transcripts and total retinal mRNA. The opsin produced is in unglycosylated (30 kDa) and two glycosylated (35 kDa and 41 kDa) forms. Incubation of the cells expressing the above proteins with 11-cis-retinal generates rhodopsin, which was purified by immunoaffinity chromatography. The purified protein shows the expected UV/visible absorption spectrum and characteristic light-dependent activation of the rod outer segment GTP binding protein. Oocytes expressing rhodopsin exhibit light-dependent ionic currents that are detected by voltage-clamp techniques.
NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.
Authors: Chattopadhyay D , Banerjee A K
Published: May 2006

The phosphoprotein (NS) of vesicular stomatitis virus is an indispensable subunit of the virion-associated RNA polymerase (L). NS consists of a highly acidic NH2-terminal domain and a basic COOH-terminal domain. Unlike the latter, the amino acid sequences of the NH2-terminal regions are highly dissimilar among different viral serotypes, although they share structural similarities. We have cloned an NS gene into the SP6 transcription vector and replaced the 5'-terminal 80% by a full-length gene for beta-tubulin, which contains an acidic COOH-terminal domain. Here we present evidence that the chimeric tubulin-NS protein is biologically active and that the acidic region in tubulin directly affects the transcription reaction. These observations indicate that NS probably functions as an activator protein in which the acidic domain stimulates transcription of the viral genes by interacting with the RNA polymerase as observed for eukaryotic cellular transcription activators.
Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes.
Authors: Blanchette-Mackie E J , Dwyer N K , Amende L M , Kruth H S , Butler J D , Sokol J , Comly M E , Vanier M T , August J T , Brady R O
Published: May 2006

Incubation of fibroblasts derived from patients with type-C Niemann-Pick disease with low density lipoprotein results in excessive intracellular accumulation of unesterified cholesterol. Cytochemical techniques revealed that this abnormal cholesterol accumulation is associated not only with a massive storage of cholesterol in lysosomes but also with a premature cholesterol enrichment of the Golgi complex. Cholesterol appeared also in the Golgi complex of some normal fibroblasts after 24 hr of low density lipoprotein loading. These findings indicate that components of the Golgi complex play a role in the intracellular translocation of exogenously derived cholesterol and that disruptions of the cholesterol transport pathway at the Golgi may, in part, be responsible for the deficiency in cholesterol utilization in type-C Niemann-Pick fibroblasts.
Presence of antibodies to ubiquitin during the autoimmune response associated with systemic lupus erythematosus.
Authors: Muller S , Briand J P , Van Regenmortel M H
Published: May 2006

Sera from patients with systemic lupus erythematosus (SLE) were shown to react with both ubiquitin and a synthetic fragment of it (residues 22-45) in an ELISA and with ubiquitin in immunoblotting experiments. Close to 80% of lupus patients possessed ubiquitin antibodies, whereas only 55% of them possessed native DNA antibodies, a marker of SLE. Less than 16% of patients with other rheumatic autoimmune diseases possessed antibodies to ubiquitin. Our results indicate that the combined measurement of antibodies to native DNA and to ubiquitin could appreciably increase the detection of SLE cases (up to 85% in our study). It is suggested that ubiquitin, a heat shock protein, could be involved in antibody formation against ubiquitin-protein conjugates present during cellular injury and that this represents a major characteristic of the autoimmune response in SLE.
Mapping of the gene encoding the beta-amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21.
Authors: Patterson D , Gardiner K , Kao F T , Tanzi R , Watkins P , Gusella J F
Published: May 2006

The gene encoding the beta-amyloid precursor protein has been assigned to human chromosome 21, as has a gene responsible for at least some cases of familial Alzheimer disease. Linkage studies strongly suggest that the beta-amyloid precursor protein and the product corresponding to familial Alzheimer disease are from two genes, or at least that several million base pairs of DNA separate the markers. The precise location of the beta-amyloid precursor protein gene on chromosome 21 has not yet been determined. Here we show, by using a somatic-cell/hybrid-cell mapping panel, in situ hybridization, and transverse-alternating-field electrophoresis, that the beta-amyloid precursor protein gene is located on chromosome 21 very near the 21q21/21q22 border and probably within the region of chromosome 21 that, when trisomic, results in Down syndrome.
Purine metabolite inosine is an adrenergic neurotrophic substance for cultured chicken sympathetic neurons.
Authors: Zurn A D , Do K Q
Published: May 2006

Purines are ubiquitous endogenous cellular metabolites that have been postulated as neurotransmitters or neuromodulators in the nervous system. Recently, we showed that a low-molecular-mass component present in liver-conditioned medium selectively enhances the adrenergic properties of dissociated chicken sympathetic neurons in culture. We report here that this substance is inosine, a purine metabolite. Indeed, analysis of the low-molecular-mass fraction of liver-conditioned medium by HPLC shows that the neurotrophic activity coelutes with and has the same absorption spectrum as inosine. Inosine increases incorporation of [3H]leucine into neuronal protein and stimulates catecholamine, but not acetylcholine, production by the sympathetic neurons in a dose-dependent fashion (half-maximal stimulation at 10(-6) M). This effect can be blocked by 5 x 10(-6) M dipyridamole, an inhibitor of nucleoside transport. Inosine therefore appears to be capable of modulating adrenergic phenotypic expression in cultured sympathetic neurons by acting via an as-yet-unknown intracellular pathway.
DNA methylation prevents transfection of genes for specific surface antigens.
Authors: Alberti S , Herzenberg L A
Published: May 2006

Sperm and trophoblast are among the few nucleated human cells that do not express HLA class I antigens. DNA methylation, which is proposed to be a tight mechanism of regulation, may be necessary to turn off these genes. We have investigated the transfectability of HLA class I genes and of the genes for the T-cell differentiation antigens Leu-1 (CD5) and Leu-2 (CD8) in mouse L cells by using human sperm cells and choriocarcinoma cell lines, tumors of trophoblastic origin, as sources of DNA. It was found that DNA from one choriocarcinoma line (JAR) does not transfect genes for HLA, Leu-1, or Leu-2 and that DNA from two other choriocarcinoma lines (BeWo and Ima) transfects only some of the surface markers. Sperm DNA transfects genes for all the surface antigens tested except Leu-1. DNA from control cells and from the line SCH transfects all the markers studied. Southern blots show that all cell types contain apparently intact genes encoding HLA, Leu-1, and Leu-2 and reveal differences in the DNA methylation patterns of genes from different sources of DNA. We treated JAR (the cell line with the lowest transfecting ability) with 5-azacytidine and obtained demethylation of its DNA. This demethylated DNA transfects genes for both HLA class I antigens and Leu-2. Further culture of JAR cells in the absence of 5-azacytidine results in remethylation of their DNA and decreased ability to transfect these surface antigens. These findings indicate that DNA methylation affects the efficiency of transfection of surface antigen genes in L cells.