bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurovirulence determinants of genetically engineered Theiler viruses.
Authors: Fu J L , Stein S , Rosenstein L , Bodwell T , Routbort M , Semler B L , Roos R P
Published: May 2006

Theiler murine encephalomyelitis viruses (TMEVs) are picornaviruses that cause enteric and neurological disease in mice. The GDVII strain and other members of the GDVII subgroup are highly virulent and cause an acute, fatal polioencephalomyelitis following intracerebral inoculation, whereas the DA stain and other members of the TO subgroup cause a persistent, demyelinating infection. We previously produced a full-length, infectious DA cDNA clone. We now describe the generation of a full-length, infectious GDVII cDNA clone and the subsequent production of intratypic chimeric cDNAs and intratypic recombinant viruses. Inoculation of the recombinant viruses into mice demonstrated that a major determinant of TMEV neurovirulence is within the GDVII 1B (capsid protein VP2)-2C coding region, most likely in the GDVII 1B (VP2)-2A coding region. Genomic sequences 5' to this region of GDVII RNA also contribute to expression of the full neurovirulence phenotype. These data demonstrate the multigenic nature of TMEV neurovirulence, as has been reported for other viruses.
Distribution of trace levels of therapeutic gallium in bone as mapped by synchrotron x-ray microscopy.
Authors: Bockman R S , Repo M A , Warrell R P , Pounds J G , Schidlovsky G , Gordon B M , Jones K W
Published: May 2006

Gallium nitrate, a drug that inhibits calcium release from bone, has been proven a safe and effective treatment for the accelerated bone resorption associated with cancer. Though bone is a target organ for gallium, the kinetics, sites, and effects of gallium accumulation in bone are not known. We have used synchrotron x-ray microscopy to map the distribution of trace levels of gallium in bone. After short-term in vivo administration of gallium nitrate to rats, trace (nanogram) amounts of gallium preferentially localized to the metabolically active regions in the metaphysis as well as the endosteal and periosteal surfaces of diaphyseal bone, regions where new bone formation and modeling were occurring. The amounts measured were well below the levels known to be cytotoxic. Iron and zinc, trace elements normally found in bone, were decreased in amount after in vivo administration of gallium. These studies represent a first step toward understanding the mechanism(s) of action of gallium in bone by suggesting the possible cellular, structural, and elemental "targets" of gallium.
Invariant measures for unipotent translations on homogeneous spaces.
Authors: Ratner M
Published: May 2006

It is shown that all Borel probability measures invariant under unipotent translations on homogeneous spaces of connected Lie groups are algebraic.
Negative and positive regulation by transcription factor cAMP response element-binding protein is modulated by phosphorylation.
Authors: Lamph W W , Dwarki V J , Ofir R , Montminy M , Verma I M
Published: May 2006

We have shown that the transcriptional activity of the protooncogene jun (c-jun) promoter is repressed by a transcription factor, the cAMP response element-binding protein (CREB). This repression can be alleviated when CREB is phosphorylated by the catalytic subunit of protein kinase A. Repression cannot be alleviated by a mutant CREB deficient in the protein kinase A phosphorylation site (M1 CREB Ser-133----Ala), suggesting that phosphorylation of CREB at this site is essential for the relief of repression. Repression by CREB requires its binding to the c-jun promoter. In NIH 3T3 cells stably expressing CREB, c-jun is no longer induced by serum, but this repression can be relieved by treatment of the cells with forskolin, an agonist of the adenylate cyclase pathway. Thus, CREB has a dual function, that of a repressor in the absence of phosphorylation and an activator when phosphorylated by protein kinase A.
N-methyl-D-aspartate receptors contribute to excitatory postsynaptic potentials of cat lateral geniculate neurons recorded in thalamic slices.
Authors: Scharfman H E , Lu S M , Guido W , Adams P R , Sherman S M
Published: May 2006

Neurons of the cat's dorsal lateral geniculate nucleus were recorded intracellularly to study the contribution of N-methyl-D-aspartate (NMDA) receptors to excitatory postsynaptic potentials (EPSPs) and low-threshold calcium spikes. EPSPs were evoked by stimulation of retinogeniculate axons in the optic tract and/or corticogeniculate axons in the optic radiations; EPSPs from both sources were similar. These EPSPs had one or two components, and the second component had several characteristics of NMDA receptor-mediated events. For example, EPSP amplitude decreased when neurons were hyperpolarized and increased when stimulus frequency was increased; these EPSPs could also be blocked reversibly by application of the selective NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). We also studied the influence of NMDA receptors on low-threshold calcium spikes, which are large, voltage- and calcium-dependent depolarizations that are often accompanied by high-frequency action potential discharge. APV blocked synaptically activated low-threshold calcium spikes, but APV had no effect on low-threshold calcium spikes that were elicited by current injection. Therefore, APV does not appear to have a direct effect on the T-type calcium channel that is involved in generation of low-threshold calcium spikes. The voltage and frequency dependence of the NMDA receptor-mediated component of the EPSPs, as well as its ability to trigger low-threshold calcium spikes, provide for complex signal processing in the lateral geniculate nucleus.
Coclosure operators and chromatic polynomials.
Authors: Ray N , Schmitt W
Published: May 2006

A theorem about closure operators on partially ordered sets is given, and applications to the counting of colorings of graphs according to partition type are derived.
Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium.
Authors: Riley B B , Barclay S L
Published: May 2006

We examined expression of the Dictyostelium cAMP phosphodiesterase (PDE) gene under conditions that alter intracellular cAMP levels during in vitro differentiation of wild-type strain V12M2 and a sporogenous derivative, HB200. In control cultures, cellular PDE activity peaked at 6 hr and declined by 8 hr, while secreted PDE activity continued to increase through 8 hr. Lowering intracellular cAMP levels with caffeine or progesterone increased cellular and secreted PDE activities 2-fold, increased stalk cell differentiation, and inhibited spore differentiation. In contrast, exposure to 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP; a membrane-permeable cAMP analog) or ammonia (which promotes intracellular cAMP accumulation in V12M2 and HB200 cells) lowered PDE activities by as much as 45%, decreased stalk cell differentiation, and increased spore differentiation. Simultaneous exposure to 8-Br-cAMP and caffeine gave intermediate PDE activities as would be expected if 8-Br-cAMP entered the cell and bypassed the caffeine-mediated block to adenylate cyclase activation. In all cases, we observed commensurate changes in developmental PDE transcript levels. The developmental time course of expression was not significantly altered by these treatments. These results suggest that the magnitude of PDE gene expression is negatively regulated by intracellular cAMP levels and provide evidence for one of the earliest changes in gene expression that is consistent with cell-type specificity. These results are discussed in terms of a bistable switch employing intracellular cAMP as a regulator of cell fate.
Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.
Authors: Sawyer J T , Doyle D
Published: May 2006

We have translated RNAs for the two rat asialoglycoprotein receptor polypeptides together in a cell-free system containing dog pancreatic microsomes and immunoprecipitated the products with antibodies that distinguish the two proteins. In this system the proteins oligomerize, as judged by their coprecipitation with either of the subunit-specific antisera. Oligomerization does not occur between subunits synthesized without microsomes or between subunits synthesized on separate microsomes mixed during detergent solubilization. Thus, oligomerization occurs within the microsomal membrane. We calculate that oligomerization proceeds with an efficiency of approximately 85%. The receptor complex appears to represent a specific oligomer because it excludes a third membrane glycoprotein synthesized in the same reaction. Oligomerization of the asialoglycoprotein receptor in vitro should provide a useful system to study the assembly of a membrane-protein complex.
Homological properties of rings of functional-analytic type.
Authors: Wodzicki M
Published: May 2006

Strong flatness properties are established for a large class of functional-analytic rings including all C*-algebras. This is later used to prove that all those rings satisfy excision in Hochschild and in cyclic homology over almost arbitrary rings of coefficients and that, for stable C*-algebras, the Hochschild and cyclic homology groups defined over an arbitrary coefficient ring k subset C of complex numbers (e.g., k = Z or Q) vanish in all dimensions.
Ligand binding to the cell surface receptor for reovirus type 3 stimulates galactocerebroside expression by developing oligodendrocytes.
Authors: Cohen J A , Williams W V , Weiner D B , Geller H M , Greene M I
Published: May 2006

Viruses utilize normal cell surface structures as attachment sites. Interaction of viral components with these structures may alter target cell growth. In the present study, the expression and function of the cell surface receptor for reovirus type 3 (Reo3R) was studied in neonatal rat optic nerve glial cultures. The Reo3R is expressed by mature oligodendrocytes and astrocytes but not by O-2A progenitor cells. It appears at an early stage of oligodendrocyte development, coincident with the O4 marker but prior to galactocerebroside or myelin basic protein. Anti-Reo3R antibodies stimulate the expression of galactocerebroside by developing oligodendrocytes. Divalent Reo3R-binding peptides are similarly active. Maximal stimulation of galactocerebroside expression occurs with treatment as short as 4 hr, consistent with a receptor-mediated process. Cell surface structures used as an attachment site by reovirus type 3 may also play a role in the regulation of oligodendrocyte differentiation.
T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1.
Authors: Linsley P S , Clark E A , Ledbetter J A
Published: May 2006

Studies using monoclonal antibodies (mAbs) have implicated the homodimeric glycoprotein CD28 as an important regulator of human T-cell activation, in part by posttranscriptional control of cytokine mRNA levels. Although the CD28 antigen has functional and structural characteristics of a receptor, a natural ligand for this molecule has not been identified. Here we show that the CD28 antigen, expressed in Chinese hamster ovary (CHO) cells, mediated specific intercellular adhesion with human lymphoblastoid and leukemic B-cell lines and with activated primary murine B cells. CD28-mediated adhesion was not dependent upon divalent cations. Several mAbs were identified that inhibited CD28-mediated adhesion, including mAb BB-1 against the B-cell activation antigen B7/BB-1 and some mAbs against major histocompatibility complex class I antigens. B7/BB-1 expression correlated closely with CD28-mediated adhesion, but class I expression did not. Transfected COS cells expressing the B7/BB-1 antigen adhered to CD28+ CHO cells; this adhesion was blocked by mAbs to CD28 and B7/BB-1. The specific recognition by CD28 of the B-cell activation antigen B7/BB-1 represents a heterophilic interaction between members of the immunoglobulin superfamily that may serve to regulate T-cell cytokine levels at sites of B-cell activation.
Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system.
Authors: Aoyama T , Nagata K , Yamazoe Y , Kato R , Matsunaga E , Gelboin H V , Gonzalez F J
Published: May 2006

A cDNA containing the full coding region of human cytochrome b5 was inserted into a vaccinia virus cDNA expression vector. Infection of human thymidine kinase-minus (TK-) 143 cells in culture with this recombinant virus resulted in production of 0.3 nmol of cytochrome b5 per mg of cell lysate protein. The expressed cytochrome had a reduced difference spectrum with a Soret peak at 424 nm, typical of pure cytochrome b5. TK- 143 cells have little detectable endogenous cytochrome b5, cytochrome P-450 (P450), and NADPH-P450 oxidoreductase. To test whether cytochrome b5 potentiated mixed-function monooxygenation in situ, these cells were coinfected with three recombinant vaccinia viruses individually carrying cDNAs encoding cytochrome b5, NADPH-P450 oxidoreductase, and P450 form IIB1. These triple-virus-infected cells were compared to cells infected with the P450IIB1 and NADPH-P450 oxidoreductase recombinant viruses with respect to P450IIB1-catalyzed monooxygenase activities. Cytochrome b5 specifically augmented the deethylation of p-nitrophenetole in microsomal membrane fractions of infected cells or when substrate was incubated directly with cells in situ. No significant increases were seen with P450IIB1-catalyzed testosterone, 7-ethoxycoumarin, or 7-pentoxyresorufin oxidations. These data demonstrate that cytochrome b5 is capable of specifically augmenting monooxygenase activities in intact cells.
Altered phenotypic expression of immunoglobulin heavy-chain variable-region (VH) genes in Alicia rabbits probably reflects a small deletion in the VH genes closest to the joining region.
Authors: Allegrucci M , Newman B A , Young-Cooper G O , Alexander C B , Meier D , Kelus A S , Mage R G
Published: May 2006

Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy-chain (lgh) locus and has a cis effect upon the expression of heavy-chain variable-region (VH) genes encoding the a2 allotype. In heterozygous a1/ali or a3/ali rabbits, serum immunoglobulins are almost entirely the products of the normal a1 or a3 allele and only traces of a2 immunoglobulin are detectable. Adult homozygous ali/ali rabbits likewise have normal immunoglobulin levels resulting from increased production of a-negative immunoglobulins and some residual ability to produce the a2 allotype. By contrast, the majority of the immunoglobulins of wild-type a2 rabbits are a2-positive and only a small percentage are a-negative. Genomic DNAs from homozygous mutant and wild-type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and lgh probes. However, when digests with infrequently cutting enzymes were analyzed by transverse alternating-field electrophoresis, the ali DNA fragments were 10-15 kilobases smaller than the wild type. These fragments hybridized to probes both for VH and for a region of DNA a few kilobases downstream of the VH genes nearest the joining region. We suggest that this relatively small deletion affects a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. These results, and the fact that the 3' VH genes rearrange early in B-cell development, indicate that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression.
DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.
Authors: Su W , Porter S , Kustu S , Echols H
Published: May 2006

The NtrC protein activates transcription of the glnA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (sigma 54-holoenzyme form). To regulate the glnA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the glnA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.
Folding and activity of hybrid sequence, disulfide-stabilized peptides.
Authors: Pease J H , Storrs R W , Wemmer D E
Published: May 2006

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a beta-turn and an alpha-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. We show that in S peptide the alpha-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure-function relationships for many biologically active sequences.
Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA.
Authors: Milsted A , Barna B P , Ransohoff R M , Brosnihan K B , Ferrario C M
Published: May 2006

We have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen has not previously been available, Expression noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because greater than 99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing us to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.
Diffusion-controlled ligand binding to spheres partially covered by receptors: an effective medium treatment.
Authors: Zwanzig R
Published: May 2006

An effective medium treatment is used to extend Berg and Purcell's theory of diffusion-controlled ligand binding to a sphere partially covered by receptors. This treatment takes into account more completely the effects of interference between receptors. The results are in excellent agreement with computer simulations by Northrup.
In vitro maturation and encapsidation of the DNA of transposable Mu-like phage D108.
Authors: Burns C M , Chan H L , DuBow M S
Published: May 2006

Mu and D108 are related, temperate, transposable coliphages with unusual modes of DNA replication (transposition) and virion DNA maturation. These double-stranded DNA genomes replicate intrachromosomally and are matured and encapsidated linked to DNA sequences flanking the dispersed, integrated phage genomes. We have developed an in vitro system that employs crude lysates prepared from cells late in the Mu lytic cycle and that is proficient for both maturation and encapsidation of D108 DNA. Different forms of phage DNA were packaged at different efficiencies, with a circular pSC101::D108cts10 plasmid being most efficient, linearized plasmid less so, and mature virion DNA a poor substrate. The addition of purified D108 Ner protein to the reaction had no effect, whereas D108 repressor (c protein) inhibited the reaction. Escherichia coli integration host factor and D108 transposase proteins exerted an inhibitory effect on circular DNA substrates but had little effect on linear DNA packaging. This in vitro system, coupled with that developed for transposition, can now be used to biochemically dissect the protein and substrate requirements of these phages' DNA maturation pathway and the nature of the molecular switch between DNA transposition and encapsidation.
Defective viral DNA ameliorates symptoms of geminivirus infection in transgenic plants.
Authors: Stanley J , Frischmuth T , Ellwood S
Published: May 2006

Nicotiana benthamiana was transformed with a single copy of a tandem repeat of subgenomic DNA B isolated from plants infected with a Kenyan isolate of the bipartite geminivirus African cassava mosaic virus. Symptoms in transformed plants were less severe than in nontransformed controls when challenged with virus or cloned DNA of Kenyan or Nigerian isolates. Symptom amelioration was associated with the mobilization and amplification of the subgenomic DNA, producing a comparable reduction in the amount of DNA specific to each genomic component. The disproportionate reduction in the levels of full-length components (DNA A, 20%; DNA B, 70%) indicates that the episomally replicating subgenomic DNA has been amplified at the expense of full-length DNA B to three times the level of the latter. Serial infection of transformants resulted in a further decrease in symptom severity and viral DNA levels. No differences were observed in the severity of symptoms or levels of viral DNA when transformants and controls were challenged with the related geminiviruses beet curly top virus and tomato golden mosaic virus, demonstrating the specific nature of the interaction. Analysis of infected tissue showed that tomato golden mosaic virus was unable to amplify the subgenomic DNA. However, since the production of subgenomic DNA is possibly a common feature of the bipartite geminiviruses, this approach might contribute to the production of plants showing increased tolerance to a number of economically important viral diseases.
Detection of individual fluorescently labeled reovirions in living cells.
Authors: Georgi A , Mottola-Hartshorn C , Warner A , Fields B , Chen L B
Published: May 2006

Reovirus serotype 1 (Lang) can be conjugated with rhodamine B or fluorescein isothiocyanate in a way that preserves viral infectivity. We have used epifluorescence microscopy to detect individual virions bound to the surface of cells and to follow in real time the early stages of reovirus infection in living cells. Following uptake of the virus into endocytic vesicles, the movement of these vesicles can be observed readily. The vesicle movement is inhibited by nocodazole or colchicine, consistent with previous findings that the movement of intracellular vesicles is often microtubule-based.
Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis.
Authors: Blaseio U , Pfeifer F
Published: May 2006

We developed vector plasmids for the transformation of Halobacterium halobium, using the replicon region from the halobacterial phage H or from the plasmid pHH1 together with a DNA fragment conferring resistance to mevinolin. H. halobium P03, a strain lacking pHH1 as well as the restriction endonuclease activity found in wild-type H. halobium, was used as the recipient strain. All H. halobium fragments tested for autonomous replication as well as the Haloferax volcanii vector pWL102 enabled stable plasmid maintenance in this strain. A frequent loss of all vectors (including pWL102) was observed in Hf. volcanii, where >90% of the mevinolin-resistant colonies obtained after transformation had lost the vector, presumably because of restriction endonuclease activity and concomitant recombination of the mevinolin resistance marker with the chromosome. The expression of gas vesicle-encoding genes (vac) was analyzed by using a 4.5-kilobase-pair (kbp) fragment containing the plasmid-encoded p-vac gene from H. halobium or an 11-kbp fragment containing the mc-vac chromosomal gene from Haloferax mediterranei for transformation experiments with H. halobium and Hf. volcanii. These experiments indicated that the mc-vac fragment contains all information necessary to synthesize gas vesicles, whereas in the case of the smaller p-vac fragment, complementation by other genes was required for a Vac+ phenotype.
Phonon representations.
Authors: Schwinger J
Published: May 2006

The gap between the nonlocalized lattice-phonon description and the localized Einstein oscillator treatment is filled by transforming the phonon Hamiltonian back to the particle variables. The particle-coordinate, normalized, wave function for the phonon vacuum state is exhibited.
Identical Mr 70,000 S6 kinase is activated biphasically by epidermal growth factor: a phosphopeptide that characterizes the late phase.
Authors: Susa M , Thomas G
Published: May 2006

Mitogenic stimulation of quiescent mouse 3T3 cells with epidermal growth factor leads to biphasic S6 kinase activation. The kinases present in both phases of the response have been purified from 32P-labeled cells and shown to contain a phosphoprotein of equivalent Mr 70,000. Chromatographic analysis of the purified S6 kinases on a Mono Q column reveals that (i) all 32P-labeled protein coelutes with S6 kinase activity, (ii) only those fractions containing S6 kinase autophosphorylate, (iii) autophosphorylation is restricted to a single Mr 70,000 protein, and (iv) the extent of autophosphorylation directly parallels the degree of S6 kinase activation. Analysis of the two autophosphorylated S6 kinases by two-dimensional tryptic phosphopeptide mapping indicates that they are the same protein. Both in vivo 32P-labeled S6 kinases contain phosphoserine and phosphothreonine but no detectable phosphotyrosine. Two-dimensional tryptic peptide maps of the in vivo 32P-labeled S6 kinases are essentially identical, except for a single qualitative change in the late-phase S6 kinase.
Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis.
Authors: Ashkenazi A , Presta L G , Marsters S A , Camerato T R , Rosenthal K A , Fendly B M , Capon D J
Published: May 2006

Infection of mononuclear cells by human immunodeficiency virus (HIV) begins with binding of the viral envelope glycoprotein, gp120, to its receptor, CD4. CD4 contains four extracellular immunoglobulin-like domains, the first of which (V1) is sufficient for HIV binding. V1 contains three sequences homologous to the antigen-complementarity-determining regions (CDR1 to -3) of immunoglobulin variable domains. While all three immunoglobulin CDRs are involved in antigen binding, only amino acids within and flanking the CDR2-like region of CD4 have been shown previously to be involved in gp120 binding. To investigate whether other regions in V1 take part in gp120 binding, we substituted alanine for each of 64 amino acids, including all of the hydrophilic residues in this domain. Mutations at four locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) markedly affected gp120 binding, but not the overall structure of V1 as probed with eight conformationally sensitive monoclonal antibodies. Thus, the gp120-binding site of CD4 is not limited to the CDR2-like sequence and consists of several discontinuous segments. Several amino acids were identified that are critical for the conformation of V1; the importance of these residues suggests some differences in the folding of this domain compared to immunoglobulin variable domains. Three amino acid substitutions were found that increase the affinity for gp120 significantly (1.7- to 2-fold individually and 4.2-fold when combined), suggesting that it may be possible to improve the HIV-blocking ability of CD4-based molecules by increasing their gp120 binding affinity.
Suppression of human immunodeficiency virus replication by ascorbate in chronically and acutely infected cells.
Authors: Harakeh S , Jariwalla R J , Pauling L
Published: May 2006

We have studied the action of ascorbate (vitamin C) on human immunodeficiency virus type 1 (HIV-1), the etiological agent clinically associated with AIDS. We report the suppression of virus production and cell fusion in HIV-infected T-lymphocytic cell lines grown in the presence of nontoxic concentrations of ascorbate. In chronically infected cells expressing HIV at peak levels, ascorbate reduced the levels of extracellular reverse transcriptase (RT) activity (by greater than 99%) and of p24 antigen (by 90%) in the culture supernatant. Under similar conditions, no detectable inhibitory effects on cell viability, host metabolic activity, and protein synthesis were observed. In freshly infected CD4+ cells, ascorbate inhibited the formation of giant-cell syncytia (by approximately 93%). Exposure of cell-free virus to ascorbate at 37 degrees C for 1 day had no effect on its RT activity or syncytium-forming ability. Prolonged exposure of virus (37 degrees C for 4 days) in the presence of ascorbate (100-150 micrograms/ml) resulted in the drop by a factor of 3-14 in RT activity as compared to a reduction by a factor of 25-172 in extracellular RT released from chronically infected cells. These results indicate that ascorbate mediates an anti-HIV effect by diminishing viral protein production in infected cells and RT stability in extracellular virions.
Recognition by the glycoprotein hormone-specific N-acetylgalactosaminetransferase is independent of hormone native conformation.
Authors: Smith P L , Baenziger J U
Published: May 2006

Some members of the glycoprotein hormone family [luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and free alpha subunit] bear unique asparagine-linked oligosaccharides with the terminal sequence SO4-Gal-NAc beta 1,4GlcNAc beta 1,2Man alpha, whereas other members [human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH)] bear predominantly oligosaccharides terminating in the sequence sialic acid alpha-Gal beta 1,4GlcNAc beta 1,2Man alpha. We previously identified an N-acetylgalactosaminetransferase (GalNAc-transferase) in bovine pituitary membranes that specifically recognizes the alpha subunit peptide and adds GalNAc to the synthetic intermediate GlcNAc2Man3GlcNAc2. In the current study we demonstrate that bLH, hCG, hCG beta, hCG alpha, and FSH alpha are recognized by the pituitary GalNAc-transferase in vitro, whereas oFSH, hFSH, and hFSH beta are not (b-, h-, and o-indicate bovine, human, and ovine). The apparent Km values for addition of GalNAc to oligosaccharides on hCG alpha and hCG beta, 13.0 and 6.2 microM, respectively, are not altered by reduction and alkylation. Thus, recognition of the peptide determinant does not require maintenance of native tertiary structural features. In the presence of the recognition determinant the Km for addition of GalNAc to the intermediate GlcNAc2Man3GlcNAc2 is reduced from 1.2-2.6 mM to less than 13 microM. hFSH is not efficiently recognized by the GalNAc-transferase due to the absence of the recognition marker on hFSH beta and some degree of masking of the recognition marker on the alpha subunit when combined with FSH beta. Since recognition is directed at primary and possibly secondary structural features, it should be possible to determine which regions of the alpha and beta subunits are responsible for the specificity of the GalNAc-transferase.
Functional and structural similarities between the inhibitory region of troponin I coded by exon VII and the calmodulin-binding regulatory region of the catalytic subunit of phosphorylase kinase.
Authors: Paudel H K , Carlson G M
Published: May 2006

A sequence homology has been noted between the carboxyl quarter of the catalytic gamma subunit of phosphorylase kinase and the region of troponin I coded by exon VII. Because this portion of troponin I contains the inhibitory region that interacts with actin and troponin C, we have examined whether the gamma subunit of phosphorylase kinase can functionally mimic troponin I by also interacting with actin and troponin C. We have found that troponin C not only activates the isolated gamma subunit of phosphorylase kinase but also binds with approximately the same affinity as calmodulin. Although actin had no effect on the activity of the gamma subunit alone, it did inhibit the activity of gamma-calmodulin and gamma-troponin C complexes. Conversely, the gamma subunit was able to inhibit actomyosin ATPase in a process that could be overcome by calmodulin. These results suggest that actin and calmodulin (or troponin C) compete for binding to the gamma subunit. Moreover, the structural and functional similarities between the gamma subunit and troponin I suggest that the gamma subunit of phosphorylase kinase may have evolved from the fusion of a protein kinase protogene with a progenitor of exon VII of troponin I.
Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.
Authors: Burton J , Goldman C K , Rao P , Moos M , Waldmann T A
Published: May 2006

Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction.
Counting dinosaurs: how many kinds were there?
Authors: Dodson P
Published: May 2006

Dinosaurs figure prominently in discussions of mass extinctions and evolutionary metrics, but their usefulness is hampered by archaic taxonomy, imprecise biostratigraphy, and imperfect preservation that bias our understanding of dinosaur diversity. A critical evaluation shows that of 540 genera and 800 species of dinosaurs proposed since 1824, 285 genera and 336 species are probably valid. Nearly half of all genera are based on a single specimen, and complete skulls and skeletons are known for only 20% of all dinosaurs. Dinosaurs are known from every continent. Countries with the greatest known diversity of dinosaurs are (in descending order) the United States, Mongolia, China, Canada, England, and Argentina; the greatest future increases may be expected from Argentina and China. Nearly half of all dinosaur genera are of latest Cretaceous age (Campanian or Maastrichtian). Estimates of the average duration of a dinosaur genus range from 5 million to 10.5 million years, with the most likely value about 7.7 million years. Dinosaurs evolved as rapidly as Cenozoic mammals. Global dinosaur diversity during the Campanian and Maastrichtian is estimated at 100 genera per stage, using a logistic model to estimate future discoveries. A model of increasing diversity and a bottleneck model compensate for the biasis in the preserved fossil record. The number of dinosaurs that have ever lived is estimated at 900-1200 genera. The fossil record of dinosaurs is presently about 25% complete. Dinosaurs disappeared in the Maastrichtian near the peak of their historic diversity.
Serum independence of transcription from the promoter of an avian retrovirus in v-src-transformed cells is a primary, intracellular effect of increased tyrosine phosphorylation.
Authors: Dutta A , Hamaguchi M , Hanafusa H
Published: May 2006

We found that transcription from the promoter in the long terminal repeat of Rous sarcoma virus in rat 3Y1 cells is dependent on the presence of serum in the culture. However, this serum dependence of transcription was relieved when 3Y1 cells were transformed by the oncogene v-src. Crossfeeding experiments showed no evidence for the production of a serum-substituting extracellular growth factor by the transformed cells. Using 3Y1 cells transformed with temperature-sensitive Rous sarcoma virus, we showed that the tyrosine kinase activity of pp60v-src was responsible for the serum-sparing effect on the level of RNA expressed from the viral promoter. Sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases that nonspecifically elevates the level of phosphotyrosine-containing proteins in cells, stimulated transcription from the viral promoter. The effects of both pp60v-src and orthovanadate were resistant to cycloheximide. These results suggest that the serum independence of transcription from the viral promoter in v-src-transformed cells was probably due to the constitutive activation of intracellular growth-factor pathways by the tyrosine kinase activity of pp60v-src.
Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.
Authors: Amemiya C T , Litman G W
Published: May 2006

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.
Interaction of epsilon-globin cis-acting control elements with erythroid-specific regulatory macromolecules.
Authors: Wu J , Grindlay G J , Johnson C , Allan M
Published: May 2006

We have used a competition assay to investigate the influence of erythroid-specific cellular factors on transcription from the human epsilon-globin major cap site promoter and the minor promoter located 200 base pairs (bp) upstream from the epsilon-globin cap site. In the human erythroid cell line K562, competition of the epsilon-globin major cap site promoter linked to the chloramphenicol acetyltransferase (CAT) gene (epsilon P-CAT) with the same promoter fragment linked to a neomycin resistance gene (epsilon P-NEO) leads to a reduction in CAT activity. This indicates the specific presence of K562 cells of factor(s) which interact with the 200-bp promoter fragment (isolated from the gene body or flanking sequences) to activate transcription from the epsilon-globin major cap site. Competition of the epsilon-globin major promoter (as epsilon P-CAT) with the upstream minor epsilon-globin promoter (as epsilon P2-NEO) also leads to a reduction in CAT activity, indicating that both promoters share erythroid-specific trans-acting factors. The reverse competition (epsilon P2-CAT with epsilon P-NEO) leads to an increase in CAT activity, suggesting that the existence of erythroid-specific factor(s) which repress transcription from the 200-bp-upstream epsilon-globin promoter.
A 170-kDa membrane-bound protease is associated with the expression of invasiveness by human malignant melanoma cells.
Authors: Aoyama A , Chen W T
Published: May 2006

Malignant spreading of cancer cells requires cell surface proteases that cleave the crosslinked collagenous matrix of connective tissues. From correlating the morphologically defined invasiveness of tumor cells with the presence of specific membrane-associated proteases, we have identified a malignant human melanoma cell line, LOX, that invades crosslinked gelatin films in vitro and contains uniquely a neutral 170-kDa gelatinase in the cell membrane. A similar gelatinase was found in membranes recovered from culture media conditioned with LOX. The 170-kDa gelatinase is a wheat germ agglutinin-binding protein. The proteolytic activity is maximal at neutral pH, enhanced by EDTA and dithiothreitol, inhibited by the cysteine protease inhibitors N-ethylmaleimide, HgCl2, and phenylmethylsulfonyl fluoride, and can bind to an organomercurial adsorbent, suggesting that it is a neutral sulfhydryl-sensitive protease. This 170-kDa gelatinase of LOX cells was not found in a control melanoma cell line, SK-MEL28, or in 32 other tumor cell lines that did not show extracellular gelatin degradation. Thus, we have identified a large membrane-bound protease that may be a specific marker molecule for melanoma cell invasiveness.
Phonon dynamics.
Authors: Schwinger J
Published: May 2006

An atomic lattice in its ground state is excited by the rapid displacement and release of an atomic constituent. The time dependence of the energy transfer to other constituents is studied by using a phonon dispersion relation that is linear in frequency and propagation vector components.
Analysis of expression of cholecystokinin in dopamine cells in the ventral mesencephalon of several species and in humans with schizophrenia.
Authors: Schalling M , Friberg K , Seroogy K , Riederer P , Bird E , Schiffmann S N , Mailleux P , Vanderhaeghen J J , Kuga S , Goldstein M
Published: May 2006

The ventral mesencephalons of hamster, guinea pig, cat, monkey, and several humans with and without the diagnosis of schizophrenia were analyzed with in situ hybridization and immunohistochemistry. Extensive codistribution of cholecystokinin mRNA and tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] mRNA was observed in cats and monkeys as well as in all five human subjects with the diagnosis of schizophrenia and in two out of five control brains. Double labeling revealed coexistence of the two markers in cat, monkey, and human. No cholecystokinin mRNA or cholecystokinin peptide was detected in the substantia nigra/ventral tegmental area of the hamster or guinea pig, even after acute and chronic neuroleptic treatment.
Molecular mapping of the mouse db mutation.
Authors: Bahary N , Leibel R L , Joseph L , Friedman J M
Published: May 2006

Diabetes (db) is an autosomal recessive mutation located in the midportion of mouse chromosome 4 that results in profound obesity with hyperphagia, increased metabolic efficiency, and insulin resistance. To clone this gene and generate a molecular map of the region around this mutation, two genetic crosses were established: an intraspecific backcross between C57BL/6J db/db females and C57BL/6J db/db x DBA/2J +/+ F1 (B6D2 db/+ F1) male mice and an interspecific intercross between B6D2 db/+ F1 males and C57BL/6J db/db x Mus spretus F1 (B6spretus db/+ F1) females. The progeny of both crosses were characterized for genotype at the db locus to map a series of restriction fragment length polymorphisms relative to the db locus. Measurements of body weight, body length, and plasma concentrations of glucose and insulin in the animals allowed the assignment of genotype (db/db vs. db/+ or +/+). A total of 132 progeny of the intraspecific cross and 48 db/db progeny of the interspecific cross were typed for individual restriction fragment length polymorphisms to generate a gene order of: centromere-brown (Mt4)-P lambda Mm3(2)-Ifa (Inta)-Cjun-db-D4Rp1-Glut1-Mtv-13-Lck. Several of the genes that are linked to db [Cjun, glucose transporter (Glut1) and Lck] map to human chromosome 1p, suggesting that db may be part of a syntenic group between human 1p and the distal portion of mouse chromosome 4. In addition, phenotyping of the progeny of these crosses revealed a wide range in plasma concentrations of glucose and insulin among the obese progeny, with some animals developing overt diabetes and other remaining euglycemic. Distributions of age-controlled plasma [glucose] and [insulin] among the intraspecific-cross obese progeny were not bimodal, suggesting a role for polygenic differences between the progenitor strains (C57BL/6J and DBA/2J) in the development of overt diabetes.