bluuebunny/crossref_metadata_2025 huggingface dataset:
The same substitution, glutamic acid----lysine at position 501, occurs in three alloalbumins of Asiatic origin: albumins Vancouver, Birmingham, and Adana.
Authors: Huss K , Madison J , Ishioka N , Takahashi N , Arai K , Putnam F W
Published: May 2006

A strategy is described for identifying structural changes in genetic variants of human serum albumin (alloalbumins). By use of this strategy we have determined an amino acid substitution in three alloalbumins of Asiatic origin. The same amino acid exchange, glutamic acid----lysine at position 501, occurs in albumins Vancouver and Birmingham, both from families that migrated from northern India, and also in albumin Adana from Turkey. This exchange corresponds to a single base mutation in the codon GAG to AAG and accords with the slow mobility of the three albumins at pH 8.6. Each of the three alloalbumins had been reported to be a new variant, yet they have the same substitution. These results emphasize the need for structural study of genetic variants that have been differentiated only by nonspecific physical criteria such as dye binding and electrophoretic mobility. We know of no other description of the substitution involved in an alloalbumin originating from the Indian subcontinent. However, the same change of glutamic acid----lysine at position 501 may be present in several other named variants reported for populations in north India and the surrounding regions.
Receptors for the host low density lipoproteins on the hemoflagellate Trypanosoma brucei: purification and involvement in the growth of the parasite.
Authors: Coppens I , Baudhuin P , Opperdoes F R , Courtoy P J
Published: May 2006

The slender bloodstream form of Trypanosoma brucei shows receptor-mediated endocytosis of low density lipoprotein (LDL) particles of its hosts. We have purified the LDL receptor of this species nearly to homogeneity (about 1000-fold purification) and obtained monospecific polyclonal antibodies against it. As analyzed by NaDodSO4/polyacrylamide gel electrophoresis, the purified receptor consists of a single subunit, with an apparent molecular mass of 86 kDa. Its isoelectric point is 5.9. On the average, each cell exposes 52,000 copies of low-affinity receptors (Kd of 250 nM) and 1800 copies of high-affinity receptors (Kd of 5.7 nM). According to indirect en bloc immunolabeling of fixed parasites, the receptor appears to be localized to the flagellar pocket membrane and the flagellar membrane and to be completely absent from the rest of the pericellular membrane. LDL is required for optimal growth of the trypanosome in vitro: cell growth can be inhibited either by removal of LDL from the culture medium or by antibodies against the purified LDL receptors. In both cases, growth is restored by the addition of excess LDL.
Three-dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells.
Authors: Isobe Y , Warner F D , Lemanski L F
Published: May 2006

The ultrastructural distribution of alpha-actinin was studied in cultured hamster heart cells by immunogold replica electron microscopy. This technique enabled us to localize alpha-actinin within the cytoskeletal networks at high resolution and in three dimensions. Colloidal gold, indicating the presence of alpha-actinin, was localized on the Z bands of nascent myofibrils in myocytes and on stress fiber bundles in nonmuscle cells. alpha-Actinin staining was also seen on stellate foci, where cytoskeletal filaments converged along the inner myocyte cell membranes. Intermediate filaments were associated with Z bands of myofibrils, stress fibers, and subplasmalemmal actin networks at the specific points where alpha-actinin was localized on these structures. Heavy meromyosin treatment prior to immunostaining confirmed that the thin filaments contained actin. These results suggest that alpha-actinin serves to interlink these various cytoskeletal elements. In addition, this protein may be involved in the initial phases of filament organization during myofibrillogenesis along the inner surface of the myocyte plasma membrane.
Organization of the human alpha 2-plasmin inhibitor gene.
Authors: Hirosawa S , Nakamura Y , Miura O , Sumi Y , Aoki N
Published: May 2006

We have isolated overlapping phage genomic clones covering an area of 26 kilobases that encodes the human alpha 2-plasmin inhibitor. The alpha 2-plasmin inhibitor gene contains 10 exons and 9 introns distributed over approximately 16 kilobases of DNA. To our knowledge, the number of introns is the highest yet reported for a member of the serine protease inhibitor (serpin) superfamily. All introns are located in the 5'-half of the corresponding mRNA. The 5'-untranslated region and the leader sequence are interrupted by 3 introns totaling approximately equal to 6 kilobases. A "TATA box" sequence is located 17 nucleotides upstream from the proposed transcription initiation site. Multiple "GC box" sequences, G + C-rich sequences, and "CCAAT box"-like sequence, the hepatitis B virus enhancer element-like sequence and the human immunodeficiency virus enhancer-like sequence appear in the 5'-flanking region. The NH2-terminal region, which implements factor XIII-catalyzed cross-linking of alpha 2-plasmin inhibitor to fibrin, is encoded by the 4th exon. The reactive site and plasminogen-binding site, both located in the COOH-terminal region, are encoded by the 10th exon. When similar amino acids of alpha 2-plasmin inhibitor and other members of the serpin gene superfamily are aligned, the position of the 7th intron of the alpha 2-plasmin inhibitor gene aligns precisely with that of the second intron of the genes for rat angiotensinogen and human alpha 1-antitrypsin genes and is misaligned by only one nucleotide with that of the third intron of antithrombin III, suggesting that the alpha 2-plasmin inhibitor gene originates from the common ancestor of these serine protease inhibitors.
Coordinate regulation of mRNAs encoding adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyltransferase by phorbol esters in human thymocytes.
Authors: Martinez-Valdez H , Cohen A
Published: May 2006

Incubation of human thymocytes in the presence of phorbol esters caused a reversible decrease in the mRNAs encoding terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) and adenosine deaminase (ADA; EC 3.5.4.4) and an increase in the mRNA encoding purine nucleoside phosphorylase (PNP; EC 2.4.2.1). The effect of phorbol esters on TdT and ADA mRNA levels can be attributed to an apparent decrease in the stability of the mRNAs. The changes in ADA, TdT, and PNP mRNAs closely simulate changes in the activities of these enzymes that occur during T-cell differentiation in vivo, suggesting a role for protein kinase C activation in the regulation of the expression of these genes during intrathymic T-cell differentiation. A role for these purine degradation enzymes in the regulation of intracellular pools of the deoxynucleotide substrates of TdT is discussed.
Subunit interactions within the T-cell antigen receptor: clues from the study of partial complexes.
Authors: Bonifacino J S , Chen C , Lippincott-Schwartz J , Ashwell J D , Klausner R D
Published: May 2006

The T-cell antigen receptor is a multisubunit complex composed of seven transmembrane chains (alpha beta gamma delta epsilon zeta 2). Subunit interactions within this complex were defined by analyzing the subunit composition of partial complexes. These partial complexes were observed in mutant and tumor T cells that fail to synthesize one or more of the receptor chains or in fibroblasts transfected with genes encoding T-cell antigen receptor chains. In addition, partial complexes were generated by immunoprecipitation with antibodies that cause selective dissociation of T-cell antigen receptor chains. The alpha and beta chains were found to form a disulfide-linked dimer in the absence of any of the other chains. The gamma, delta, and epsilon chains were also efficiently associated in the absence of a complete heterodimer. Complexes composed only of delta epsilon or gamma epsilon could be observed. Both these dimers, as well as the gamma delta epsilon trimer, could form stable complexes with alpha beta, even in the absence of zeta 2. The zeta 2 dimer could bind directly to alpha beta. In the absence of a complete clonotypic heterodimer, zeta 2 was not stably associated with gamma delta epsilon. These observations suggest a model in which alpha beta interacts directly with the gamma delta epsilon trimer and zeta 2, with less-direct interaction between the latter two.
Genetic exchange among natural isolates of bacteria: recombination within the phoA gene of Escherichia coli.
Authors: DuBose R F , Dykhuizen D E , Hartl D L
Published: May 2006

An 1871-nucleotide region including the phoA gene (the structural gene encoding alkaline phosphatase, EC 3.1.3.1) was cloned and sequenced from eight naturally occurring strains of Escherichia coli. Alignment with the sequence from E. coli K-12 made apparent that there were 87 polymorphic nucleotide sites, of which 42 were informative for phylogenetic analysis. Maximum parsimony analysis revealed six equally parsimonious trees with a consistency index of 0.80. Of the 42 informative sites, 22 were inconsistent with each of the maximum parsimony trees. The spatial distribution of the inconsistent sites was highly nonrandom in a manner implying that intragenic recombination has played a major role in determining the evolutionary history of the nine alleles. The implication is that different segments of the phoA gene have different phylogenetic histories.
Relativistic cyclotron resonance shape in magnetic bottle geonium
Authors: Dehmelt Hans , Mittleman Richard , Liu Yuan
Published: May 2006

The thermally excited axial oscillation of the electron through the weak magnetic bottle needed for the continuous Stern-Gerlach effect modulates the cyclotron frequency and produces a characteristic ≈ 12-kHz-wide vertical rise-exponential decline line shape of the cyclotron resonance. At the same time the relativistic mass shift decreases the frequency by ≈ 200 Hz per cyclotron motion quantum level
n
. Nevertheless, our analysis of the complex line shape shows that it should be possible to produce an abrupt rise in the cyclotron quantum number
n
from 0 to ≈ 20 over a small fraction of 200 Hz, when the 160-GHz microwave drive approaches the
n
= 0 → 1 transition, and a jump of 14 levels over a frequency increment of 200 Hz has already been observed in preliminary work. This realizes an earlier proposal to generate a very sharp cyclotron resonance feature by quasithermal excitation with a square noise band and should provide a way to detect spin flips when a weak bottle is used to reduce the broadening of the
g
- 2 resonance by a factor of 20.
Molecular cloning, sequencing, and mapping of EGR2, a human early growth response gene encoding a protein with "zinc-binding finger" structure.
Authors: Joseph L J , Le Beau M M , Jamieson G A , Acharya S , Shows T B , Rowley J D , Sukhatme V P
Published: May 2006

Early growth response gene-1 (Egr-1) is a mouse gene displaying fos-like induction kinetics in diverse cell types following mitogenic stimulation. Egr-1 encodes a protein with "zinc-binding finger" structure. Zinc fingers are a protein structural motif that serve as DNA-binding domains in several transcriptional regulatory proteins. Using low-stringency hybridization with an Egr-1 cDNA probe, we identified a distinct human cDNA (designated EGR2 for early growth response gene-2), which is coregulated with EGR1 by fibroblast and lymphocyte mitogens; however, several stimuli that induce Egr-1 mRNA in PC12 (rat pheochromocytoma) cells do not induce Egr-2 mRNA. The cDNA sequence predicts a protein of 406 amino acids, including three tandem zinc fingers of the Cys2-His2 class. Strikingly, the deduced amino acid sequences of human EGR2 and mouse Egr-1 are 92% identical in the zinc finger region but show no similarity elsewhere. EGR2 maps to human chromosome 10 at bands q21-22. Structure-function analysis of EGR2 and EGR1 proteins should provide insight into the mechanisms linking signal transduction and transcriptional regulation of gene expression.
Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.
Authors: Wexler I D , Kerr D S , Ho L , Lusk M M , Pepin R A , Javed A A , Mole J E , Jesse B W , Thekkumkara T J , Pons G
Published: May 2006

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.
5,7-Dihydroxytryptamine identifies living dopaminergic neurons in mesencephalic cultures.
Authors: Silva N L , Mariani A P , Harrison N L , Barker J L
Published: May 2006

The autofluorescent serotonin analogue 5,7-dihydroxytryptamine (5,7-DHT) was used to identify living catecholaminergic neurons in monolayer cultures derived from the embryonic rat mesencephalon. A high correlation between 5,7-DHT accumulation and aldehyde-induced catecholamine fluorescence as well as tyrosine hydroxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase immunoreactivity was found. This indicates that these cells were dopamine-containing neurons. Whole-cell patch recordings showed that all mesencephalic neurons had resting membrane potentials of -50 mV or greater and input resistances ranging between 200 and 700 M omega and exhibited spontaneous action potentials and postsynaptic potentials. The duration of the action potential of the dopamine-containing neurons was characteristically longer than that of the non-dopamine-containing mesencephalic cells. In some dopamine-containing neurons, repolarization of the action potential was clearly biphasic, and the slow phase of repolarization was reversibly blocked by local application of Cd2+ or Co2+. This "shoulder" in the action potential was never observed in non-dopamine-containing neurons, where Cd2+ or Co2+ application was always without effect. It is concluded that 5,7-DHT can be used to identify living dopamine-containing neurons in dissociated mesencephalic cultures and these neurons express distinct electrical properties.
Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction.
Authors: Matteoli M , Haimann C , Torri-Tarelli F , Polak J M , Ceccarelli B , De Camilli P
Published: May 2006

The regulatory peptide called calcitonin gene-related peptide (CGRP) was detected by immunofluorescence in frog motor neurons and motor nerve terminals. In motor nerve terminals, CGRP-like immunoreactivity was found to be segregated within large dense-core vesicles. To determine whether exocytosis from acetylcholine-containing small synaptic vesicles and from CGRP-containing large dense-core vesicles can be independently stimulated, nerve-muscle preparations were exposed to alpha-latrotoxin. This toxin induced complete depletion of acetylcholine-containing small synaptic vesicles but did not induce a parallel depletion of CGRP-like immunoreactivity and of large dense-core vesicles. These effects were independent of the presence of extracellular Ca2+ and occurred both at room temperature and at low temperature (1-3 degrees C). These findings suggest that exocytosis from the two vesicle populations is mediated by distinct biochemical mechanisms, which might be differentially regulated by physiological stimuli.
Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector.
Authors: Rekosh D , Nygren A , Flodby P , Hammarskjöld M L , Wigzell H
Published: May 2006

A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium. Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.
Immortalization of human lymphocytes by transfection with DNA from mouse L929 cytoplasts.
Authors: Abken H , Bützler C , Willecke K
Published: May 2006

Transfection of human peripheral blood lymphocytes with DNA from mouse L929 cytoplasts induced proliferation of lymphocytes and the formation of B and T cell-derived cell lines with apparently unlimited growth potential. The cell lines could be grown in serum-containing media as well as in chemically defined serum-free media, have a nearly normal human karyotype, did not form colonies in soft-agar medium, and were not tumorigenic after injection into nude mice. For immortalization of human lymphocytes, DNA from L929 cytoplasts was 100-fold more efficient than L929 nuclear DNA. The ability of cytoplast DNA to immortalize lymphocytes could be consecutively transferred by using total cellular DNA from primary or secondary transfectants. Circular or linear mitochondrial DNA of L929 cells did not lead to immortalization of human lymphocytes. Since DNA with immortalizing activity could be isolated from cytoplasts, the Hirt supernatant, and a mitochondria-depleted cytoplasmic fraction of L929 cells, we conclude that the immortalizing DNA is located extramitochondrially in the cytoplasm of L929 cells.
Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling.
Authors: Novick S L , Hoekstra D
Published: May 2006

The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.
Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates.
Authors: Sarin P S , Agrawal S , Civeira M P , Goodchild J , Ikeuchi T , Zamecnik P C
Published: May 2006

Antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages were examined for their ability to inhibit human immunodeficiency virus (HIV)-induced syncytium formation and virus expression. HIV inhibitory activity was found to be dependent on both chain length and the number of phosphonate residues. Introduction of 18 phosphonate groups in an oligomer of chain length 20 significantly increased HIV inhibitory activity relative to the parent oligonucleotide, whereas 5 such groups showed little or no increase in the HIV inhibition capacity. Methylphosphonate-linked oligomers are more stable to nuclease degradation and hence could be potentially useful in the treatment of acquired immunodeficiency syndrome.
Adhesive receptor Mac-1 coordinates the activation of factor X on stimulated cells of monocytic and myeloid differentiation: an alternative initiation of the coagulation protease cascade.
Authors: Altieri D C , Morrissey J H , Edgington T S
Published: May 2006

Monocytes initiate coagulation through regulated surface expression of tissue factor and local assembly of a proteolytic enzymatic complex formed by tissue factor and factor VII/activated factor VII. We now show that, in the absence of these initiating molecules, monocytes and cell lines of monocytic/myeloid differentiation can alternatively initiate coagulation after exposure to ADP. The molecular basis for this procoagulant response consists of two distinct events. First, cell stimulation with ADP induces high-affinity binding of coagulation factor X to the surface-adhesive receptor Mac-1. Locally, Mac-1-concentrated factor X is then rapidly proteolytically cleaved to an active protease with size and activity characteristics of activated factor X, which supports the cell-associated formation of thrombin and the procoagulant response. We conclude that the monocytic/myeloid adhesive receptor Mac-1 has the unexpected, specifically inducible property to organize a molecular assembly culminating in rapid fibrin formation that is independently regulated from tissue factor and factor VII/activated factor VII.
Genetic determinants of neoplastic transformation by the retroviral oncogene v-erbB.
Authors: Wells A , Bishop J M
Published: May 2006

The retroviral oncogene v-erbB is a mutant version of the gene (c-erbB or ERBB1) that encodes the cell-surface epidermal growth factor receptor (EGFR). The mutations take three forms: (i) a large deletion that removes the entire ligand-binding domain of EGFR, (ii) smaller deletions that affect the carboxyl-terminal domain of EGFR, and (iii) point mutations that cause conservative substitutions of amino acids. Previous work has shown that, in the absence of the large deletion, ERBB1 cannot transform cells autonomously. Here we report that when the large deletion is present, no other mutation is required for ERBB1 to transform established rodent fibroblasts to a tumorigenic phenotype. In particular, there is no need for deletions affecting the carboxyl terminus of the gene product. It appears, therefore, that removal of the ligand-binding domain from the EGFR suffices to create a transforming protein. Deletions at the carboxyl terminus of the EGFR apparently play only a secondary role in transformation by affecting the host range and perhaps the potency of transformation; and there is as yet no evidence to implicate point mutations in the activation of ERBB1 to an oncogene. Our findings support the view that augmented activity of the EGFR can contribute to tumorigenesis.
Cloning and characterization of a cDNA encoding the chloroplastic copper/zinc-superoxide dismutase from pea.
Authors: Scioli J R , Zilinskas B A
Published: May 2006

A cDNA encoding the chloroplastic copper/zinc-superoxide dismutase of pea (Pisum sativum L.) was isolated from a cDNA library constructed in lambda gt11 from leaf mRNA. Nucleotide sequence analysis of the 875-base-pair clone revealed that it contained the complete coding sequence of the mature superoxide dismutase isozyme subunit, along with sequence information for a 48-amino acid N-terminal transit peptide. The deduced amino acid sequence of the mature subunit proved to be 64-87% homologous with amino acid sequences of copper/zinc-superoxide dismutases from other plant species. In vitro transcription, followed by cell-free translation, of the cDNA resulted in the formation of a 23.5-kDa precursor polypeptide, which, upon incubation with isolated pea chloroplasts, was imported and processed to its mature subunit molecular mass of 17.4 kDa.
Human ETS2 gene on chromosome 21 is not rearranged in Alzheimer disease.
Authors: Sacchi N , Nalbantoglu J , Sergovich F R , Papas T S
Published: May 2006

The human ETS2 gene, a member of the ETS gene family, with sequence homology with the retroviral ets sequence of the avian erythroblastosis retrovirus E26 is located on chromosome 21. Molecular genetic analysis of Down syndrome (DS) patients with partial trisomy 21 allowed us to reinforce the supposition that ETS2 may be a gene of the minimal DS genetic region. It was originally proposed that a duplication of a portion of the DS region represents the genetic basis of Alzheimer disease, a condition associated also with DS. No evidence of either rearrangements or duplications of ETS2 could be detected in DNA from fibroblasts and brain tissue of Alzheimer disease patients with either the sporadic or the familiar form of the disease. Thus, an altered ETS2 gene dosage does not seem to be a genetic cause or component of Alzheimer disease.
Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (beta 2-microglobulin) chain genes in transgenic mice.
Authors: Chamberlain J W , Nolan J A , Conrad P J , Vasavada H A , Vasavada H H , Ploegh H L , Ganguly S , Janeway C A , Weissman S M
Published: May 2006

We introduced the human genes HLA-B7 and B2M encoding the heavy (HLA-B7) and light [beta 2-microglobulin (beta 2m)] chains of a human major histocompatibility complex class I antigen into separate lines of transgenic mice. The tissue-specific pattern of HLA-B7 RNA expression was similar to that of endogenous class I H-2 genes, although the HLA-B7 gene was about 10-fold underexpressed in liver. Identical patterns of RNA expression were detected whether the HLA-B7 gene contained 12 or 0.66 kilobase(s) (kb) of 5' flanking sequence. The level of expression was copy number dependent and as efficient as that of H-2 genes; gamma interferon enhanced HLA-B7 RNA expression in parallel to that of H-2. In addition to the mechanism(s) responsible for gamma interferon-enhanced expression, there must be at least one other tissue-specific mechanism controlling the constitutive levels of class I RNA. Tissue-specific human beta 2m RNA expression was similar to that of mouse beta 2m, including high-level expression in liver. Cell surface HLA-B7 increased 10- to 17-fold on T cells and on a subset of thymocytes from HLA-B7/B2M doubly transgenic mice compared to HLA-B7 singly transgenic mice. The pattern of expression of HLA-B7 on thymocytes resembled that of H-2K as opposed to H-2D. These results confirm that coexpression of both human chains is required for efficient surface expression and that HLA-B7 may share a regulatory mechanism with H-2K, which distinguishes it from H-2D.
Thymotaxin: a thymic epithelial peptide chemotactic for T-cell precursors.
Authors: Imhof B A , Deugnier M A , Girault J M , Champion S , Damais C , Itoh T , Thiery J P
Published: May 2006

The embryonic thymus is seeded by invading hemopoietic precursor cells that differentiate intrathymically into T lymphocytes. We have recently reported that avian thymic epithelial cells secrete chemotactic peptides, which provoke oriented migration of hemopoietic precursor cells in vitro. The established rat thymic epithelial cell line IT-45 R1 produced a polypeptide that resolves as a single band in the region of 11 kDa on NaDodSO4/polyacrylamide gels. This molecule, which we have named thymotaxin, induced a chemotactic response in a subpopulation of hemopoietic cells from juvenile rat bone marrow. Responding cells were generated by short-term coculture of rat bone marrow hemopoietic cells with mouse bone marrow stroma in a steroid-free medium. Cells selected in a chemotactic chamber have a lymphoid or blast cell morphology. The phenotype of the responding cells is Thy-1+, CD4- [corrected] and CD8-. In contrast, CD8 T-lymphocyte differentiation antigen was expressed after coculture with embryonic thymic monolayers, suggesting that the responding cells correspond to the precursors colonizing the thymus.
Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen.
Authors: Wright S D , Weitz J I , Huang A J , Levin S M , Silverstein S C , Loike J D
Published: May 2006

Human polymorphonuclear leukocytes (PMN) have previously been shown to bind to aggregates of fibrin and to fibrinogen-coated surfaces. During their interactions with fibrinogen-coated surfaces, PMN make such close contact with the surface that a portion of the secreted elastase activity is protected from macromolecular protease inhibitors in the surrounding medium. Here we show that the receptor on PMN that mediates this interaction is complement receptor type 3 (CR3; CD11b/CD18), a molecule previously identified as a receptor for the complement protein fragment C3bi. Monoclonal antibodies against CR3 that block the binding of C3bi also block the binding of PMN to fibrinogen-coated surfaces and the formation of a protected compartment. The region of fibrinogen recognized by CR3 lies at the carboxyl terminus of the gamma chain, since peptides based on this sequence effectively inhibit the binding of PMN to fibrinogen-coated surfaces. These peptides also block the binding of C3bi-coated erythrocytes to CR3, thus indicating that a single binding site is used for binding both C3bi and fibrinogen. Sequence analysis shows strong structural similarity between this region of fibrinogen and other known ligands of CR3. These studies thus indicate that CR3 functions as a receptor not only for C3bi but also for fibrinogen.
Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase.
Authors: Szewczyk B , Laver W G , Summers D F
Published: May 2006

The virion-associated RNA polymerase and the structural nucleoprotein of influenza A virus were separated by sodium dodecyl sulfate/PAGE, electroblotted to a polyvinylidine membrane, and eluted with good recovery from the membrane. After renaturation by incubating with Escherichia coli thioredoxin, these proteins were active in a reconstituted in vitro transcription reaction with purified genomic RNAs. All four proteins (i.e., the three subunits of the RNA polymerase as well as the structural nucleoprotein) were required for activity. The RNA products were plus-strand, mRNA-sized species.
Negative and positive factors determine the activity of the polyoma virus enhancer alpha domain in undifferentiated and differentiated cell types.
Authors: Wasylyk B , Imler J L , Chatton B , Schatz C , Wasylyk C
Published: May 2006

The host range of polyoma virus is dependent upon the activity of its enhancer, which is inactive in undifferentiated embryonal carcinoma cells, such as F9 cells, and is active after their differentiation. We show here that the activity of the alpha domain of the polyoma virus enhancer displays a similar cell-specificity and inducibility as does the whole enhancer. We present evidence to show that its activity is determined by the balance between the activities of two factors, PEA2, a labile repressor, and PEA1, an inducible positive factor that we have characterized previously. Changes in repressor activity help account for the increase in alpha-domain activity after differentiation of F9 cells. These results suggest that PEA2 is crucial in the regulation of viral gene expression and perhaps more generally in the control of gene expression during differentiation.
Maculosin, a host-specific phytotoxin for spotted knapweed from
Alternaria alternata
Authors: Stierle Andrea C. , Cardellina John H. , Strobel Gary A.
Published: May 2006

Several diketopiperazines have been isolated from liquid cultures of
Alternaria alternata
, the causal agent of black leaf blight of spotted knapweed,
Centaurea maculosa
Lam. One of these compounds, maculosin [the diketopiperazine cyclo(-L-Pro-L-Tyr-)], was active in the nicked-leaf bioassay at 10
-5
M; synthetic maculosin possessed chemical and biological activities identical to those of the natural product. Other diketopiperazines isolated from the fungus possessed either less activity or none at all. In tests against 19 plant species, maculosin was phytotoxic only to spotted knapweed. Thus maculosin is a host-specific phytotoxin from a weed pathogen.
Axonal delay lines for time measurement in the owl's brainstem.
Authors: Carr C E , Konishi M
Published: May 2006

Interaural time difference is an important cue for sound localization. In the barn owl (Tyto alba) neuronal sensitivity to this disparity originates in the brainstem nucleus laminaris. Afferents from the ipsilateral and contralateral magnocellular cochlear nuclei enter the nucleus laminaris through its dorsal and ventral surfaces, respectively, and interdigitate in the nucleus. Intracellular recordings from these afferents show orderly changes in conduction delay with depth in the nucleus. These changes are comparable to the range of interaural time differences available to the owl. Thus, these afferent axons act as delay lines and provide anatomical and physiological bases for a neuronal map of interaural time differences in the nucleus laminaris.
Intracerebral xenografts of human mesencephalic tissue into athymic rats: immunochemical and in vivo electrochemical studies.
Authors: Strömberg I , Almqvist P , Bygdeman M , Finger T E , Gerhardt G , Granholm A C , Mahalik T J , Seiger A , Hoffer B , Olson L
Published: May 2006

Intracerebral allografts of fetal neurons have been studied in both rodents and nonhuman primates. Such research has been directed towards problems in developmental neurobiology and in animal models of neurological diseases. Whether intracerebrally transplanted human fetal neurons are capable of forming synapses and releasing neurotransmitters are key questions in any application of this approach to human brain development and dysfunction. We studied these questions by examining the immunocytochemical and in vivo electrochemical properties of xenografts of human mesencephalic dopaminergic neurons placed into athymic "nude" rats. The transplanted neurons survive, continue to express human-specific Thy-1 immunoreactivity, and extend neuronal processes into the host brain where morphologically identifiable synapses form. Potassium-evoked release of monoamines occurs in the vicinity of the graft but is absent in more remote areas of the host neuropil. These results indicate that human fetal tissue fragments can provide a source of viable neuroblasts for transplantation. Further, synapses form between pre- and postsynaptic elements expressing different species-specific cell surface markers; thus, these markers do not play a determining role in synaptogenesis.
c-fos promoter trans-activation by the tax1 protein of human T-cell leukemia virus type I.
Authors: Fujii M , Sassone-Corsi P , Verma I M
Published: May 2006

To understand the mechanisms of oncogenesis by human T-cell leukemia virus type I, we have investigated the ability of the tax1, protein to modulate transcription of protooncogenes. By using a transient cotransfection assay, we report that the protooncogene fos promoter is transactivated by tax1 in a variety of cell types. Two regions containing upstream sequences between positions -362/-324 and -323/-276 of the c-fos promoter responded to this activation and also conferred tax1 responsiveness to the heterologous herpesvirus thymidine kinase promoter. These two sequences include elements mediating the induction by v-sis-conditioned medium and serum, phorbol ester, or epidermal growth factor, respectively. Furthermore, expression of the endogenous c-fos gene was activated by tax1 in human T-cell leukemia virus type I-infected cell lines. In contrast, no trans-activation of the c-myc or c-Ha-ras promoter was observed.
Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis.
Authors: Calman A F , Peterlin B M
Published: May 2006

The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors. We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3. These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes. Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes. HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other. Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1. We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter. However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences. Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription. Alternatively, TF-X1 may not be a DNA-binding protein at all.
Efficient transactivation and morphologic transformation by bovine papillomavirus genes expressed from a bovine papillomavirus/simian virus 40 recombinant virus.
Authors: Settleman J , DiMaio D
Published: May 2006

To efficiently introduce bovine papillomavirus type 1 genes into cultured cells, we constructed a hybrid viral genome in which the simian virus 40 early region is replaced with a segment of the bovine papillomavirus type 1 transforming region. High-titer stocks of simian virus 40 virions containing the recombinant genome were produced in monkey cells that express simian virus 40 large tumor antigen. Cells infected with this virus efficiently expressed the bovine papillomavirus type 1 E2 and E5 genes. Expression of the E2 gene caused transactivation of genes linked to the bovine papillomavirus type 1 control region, resulting in up to a 1000-fold induction. At high multiplicity of infection of a cell line containing an integrated reporter gene, most cells were infected and responded to transactivation. Within 48 hr of infection with wild-type virus but not with an open reading frame E5 mutant, mouse C127 cells displayed dramatic changes in morphology and growth characteristics similar to those seen in tumorigenic transformation. This system can be used to determine the acute cellular response to introduction of bovine papillomavirus type 1 transforming and regulatory genes; it can also be used to induce foreign genes stably incorporated into cultured mammalian cells.
Evidence for expression of the facilitated glucose transporter in rat hepatocytes.
Authors: Rhoads D B , Takano M , Gattoni-Celli S , Chen C C , Isselbacher K J
Published: May 2006

The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment.
Vertex representations of quantum affine algebras
Authors: Frenkel Igor B. , Jing Naihuan
Published: May 2006

We construct vertex representations of quantum affine algebras of ADE type, which were first introduced in greater generality by Drinfeld and Jimbo. The limiting special case of our construction is the untwisted vertex representation of affine Lie algebras of Frenkel-Kac and Segal. Our representation is given by means of a new type of vertex operator corresponding to the simple roots and satisfying the defining relations. In the case of the quantum affine algebra of type
A
, we introduce vertex operators corresponding to all the roots and determine their commutation relations. This provides an analogue of a Chevalley basis of the affine Lie algebra [unk](
n
) in the basic representation.
A free-energy perturbation study of the binding of methotrexate to mutants of dihydrofolate reductase.
Authors: Singh U C , Benkovic S J
Published: May 2006

The importance of hydrophobic residues to the binding of methotrexate in the active site of dihydrofolate reductase (EC 1.5.1.3) was examined by a free-energy perturbation method. The replacement of a strictly conserved residue, Phe-31, by tyrosine or valine costs 1.8 and 5.1 kcal/mol, respectively, to the binding of the drug (1 cal = 4.184 J). In the case of the Phe31----Tyr mutation, the loss of the binding energy is due to the desolvation of the phenolic group; in the case of Phe31----Val mutation, it is mainly due to the loss of the interaction with the drug. The replacement of Leu-54 by glycine decreases the binding energy by 4.0 kcal/mol. A calculation on the mutation of Phe-31 to serine shows that the alteration could reduce the binding energy of methotrexate by 9.7 kcal/mol. The calculations clearly show that the hydrophobic interactions are as important as the hydrophilic ones in the binding of methotrexate.
Genetic analysis of the dominant white-spotting (W) region on mouse chromosome 5: identification of cloned DNA markers near W.
Authors: Geissler E N , Cheng S V , Gusella J F , Housman D E
Published: May 2006

We have assigned several mouse cDNA and genomic clones to the W region of mouse chromosome 5, established their position with respect to various marker loci in the region, and provided molecular verification that the W19H mutation is a deletion. Meiotic recombination analysis of an interspecific mouse backcross indicated the following gene order and distances [in centimorgans (cM)]: centromere-Emv-1-(13 cM)-D4S76-(17 cM)-D5SC25-(5 cM)-alpha-casein-(1 cM)-beta- casein-(6 cM)-alpha-fetoprotein-(18 cM)-beta-glucuronidase. D5SC25, an anonymous locus defined by a mouse brain cDNA, maps near the map position of W and within the breakpoints of the presumed genetic deletion that causes the W19H phenotype. Southern analysis of DNAs of W19H/+ interspecific F1 hybrid mice and somatic cell hybrid lines carrying the W19H deletion chromosome showed the deletion of D5SC25. In fact, analysis of other mutations at or near the W locus, which had been transferred from the strain of origin for many backcross generations, revealed the retention of donor restriction fragment length polymorphisms at the D5SC25 locus. Such evidence confirms close linkage between D5SC25 and W (within 1 cM) and indicates that the D5SC25 cDNA clone could serve as a starting point in a chromosome "walk" to W and other closely linked loci that affect development.
Opsin exhibits cGMP-activated single-channel activity.
Authors: Clack J W , Stein P J
Published: May 2006

Low concentrations of cGMP evoked reversible single-channel currents in patches excised from liposomes that contained purified bovine opsin. Two elementary conductances, of 32 and 17 pS, were observed in the presence of 10-200 microM cGMP. Both individual channel openings (mean open times, approximately 1.6 ms for the 32-pS conductance and approximately 1.0 ms for the 17-pS conductance) and bursts of openings (mean burst duration, approximately 2-3 ms for the large events) were observed. The cGMP-activated channel activity could be observed in the presence or absence of Ca2+. These results raise the possibility that opsin or rhodopsin, an opsin/rhodopsin isoform, or an opsin/isoform multimer serves as a cGMP-modulated pore in the rod outer segment.