bluuebunny/crossref_metadata_2025 huggingface dataset:
Two major classes in the M protein family in group A streptococci.
Authors: O'Toole P , Stenberg L , Rissler M , Lindahl G
Published: May 2006

The M protein family of molecules in the group A streptococcus comprises a number of cell surface proteins that interact with the immune system of the host. One of the proteins in this family is the IgA receptor Arp4, which has C repeats similar to those that characterize the known M proteins. The streptococcal strain expressing Arp4 also expresses a second immunoglobulin-binding protein, Mrp4, which is shown here to be encoded by a gene located immediately upstream of the gene for Arp4. In addition to binding IgG, Mrp4 also binds fibrinogen, a property ascribed to M proteins. DNA sequence analysis demonstrated that the Mrp4 protein indeed is a member of the M protein family, but it was unexpectedly found to have a type of repeat that is identical to the A repeat described for FcRA76, a partially sequenced streptococcal Fc receptor. Purified FcRA76 was shown to bind fibrinogen and IgG, like Mrp4. These data show that the known molecules in the M protein family can be divided into two classes, A and C, according to the type of repeat region found. Hybridization studies with a panel of clinical isolates indicate that many streptococcal strains express class A and class C proteins, whereas some strains express only class C proteins. Class A molecules show amino-terminal sequence variation, like class C molecules, which suggests that proteins of both classes are targets for the immune response.
Mutation creates an open reading frame within the 5' untranslated region of macaque erythrocyte carbonic anhydrase (CA) I mRNA that suppresses CA I expression and supports the scanning model for translation.
Authors: Bergenhem N C , Venta P J , Hopkins P J , Kim H J , Tashian R E
Published: May 2006

A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes.
High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.
Authors: Carruthers V B , Cross G A
Published: May 2006

This report describes a method for growing both bloodstream- and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HMI-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 10(6) cells at maturity. Colonies were transferred to filters and probed for multicopy and single-copy genes. Potential uses of this method in conjunction with classical and reverse genetic approaches to studying trypanosomes are discussed.
Improbability of effective vaccination against human immunodeficiency virus because of its intracellular transmission and rectal portal of entry.
Authors: Sabin A B
Published: May 2006

The worldwide effort to produce a vaccine against AIDS continues to disregard the fact that even human immunodeficiency virus (HIV)-specific neutralizing antibodies and cell-mediated immunity are ineffective against virus within cells without viral antigens on the cell membrane--and that much of HIV infection is transmitted in this manner. According to a recent report, a simian immunodeficiency virus vaccine that protected monkeys against an intravenous challenge with cell-free virus was, as predicted, ineffective against an intravenous challenge with the same amount of virus in infected cells. Moreover, antibody and HIV have been found to coexist in cell-free plasma from asymptomatic and symptomatic patients. Excluding direct introduction of HIV into the blood-stream, the most common and efficient form of transmission of HIV infection is by receptive anal intercourse, and semen contains large numbers of infected cells per milliliter. Recent reports showing that colorectal cells can be persistently infected by HIV and that HIV RNA and cDNA are present in the cells of the colon of dead AIDS patients indicate that either cell-free or intracellular HIV has the capacity to multiply at the portal of entry in the colorectal area without interference from neutralizing antibodies. The available data provide no basis for testing any HIV vaccine in human beings either before or after infection. The main challenge is to find a way to kill cells with chromosomally integrated HIV cDNA without harming normal cells, perhaps by identifying repressor proteins that might be produced by the cells with integrated HIV cDNA and thus could become specific targets for cell-killing drugs.
Puzzling questions about excited superdeformed rotational bands of atomic nuclei are answered by the two-revolving-cluster model.
Authors: Pauling L
Published: May 2006

The two-revolving-cluster model provides explanations of several questions about excited superdeformed bands: restriction to the lanthanons and the Hg-Tl-Pb region and to the smaller values of the neutron number for each element, truncation of the gamma-ray cascades, differences in shape of the lanthanon and Hg-Tl-Pb bands, alignment of quantified spins, and the existence of pairs of bands with nearly identical gamma-ray sequences. A previously unrecognized kind of pairing (intercalation of gamma-ray values) is also reported and a discussion is given of the values of electric quadrupole moments.
Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.
Authors: Adams R M , Soriano H E , Wang M , Darlington G , Steffen D , Ledley F D
Published: May 2006

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.
Protein stitchery: design of a protein for selective binding to a specific DNA sequence.
Authors: Park C , Campbell J L , Goddard W A
Published: May 2006

We present a general strategy for designing proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to establish that the dimer binds specifically to the DNA sequence TCATCGATGA but does not bind to the wild-type DNA sequences, nor to oligonucleotides in which the recognition half-site is modified by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.
Coat protein gene duplication in a filamentous RNA virus of plants.
Authors: Boyko V P , Karasev A V , Agranovsky A A , Koonin E V , Dolja V V
Published: May 2006

Computer-assisted analysis revealed a striking sequence similarity between the putative 24-kDa protein (p24) encoded by open reading frame (ORF) 5 of beet yellows closterovirus and the coat protein of this virus encoded by the adjacent ORF6. Both of these proteins are closely related to the homologous proteins of another closterovirus, citrus tristeza virus. It is hypothesized that the genes for coat protein and its diverged tandem copy have evolved by duplication. Phylogenetic analysis using various methods for tree generation suggested that the duplication was already present in the genome of the common ancestor of the two closteroviruses. The genes for p24 and coat protein of beet yellows closterovirus were cloned, transcribed, and translated in vitro yielding products of the expected size. It was shown that p24 is translated starting from the first of the two alternative AUG codons located near the 5' terminus of ORF5. The presence of a single protein species in beet yellows closterovirus virions and the near identity of the amino acid composition of this protein with the composition of the ORF6 but not the ORF5 product indicated that p24 is not a major virion component. Most of the amino acids that are conserved in the coat proteins of filamentous viruses of plants are retained also in p24. These observations suggest that p24 may share some structural and functional features with the coat protein but probably fulfills a distinct function in virus reproduction.
Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in a nonhepatic cell line.
Authors: Thrift R N , Drisko J , Dueland S , Trawick J D , Davis R A
Published: May 2006

To explore the process of lipoprotein assembly, plasmids encoding truncated forms of apolipoprotein B (apoB) were transfected into Chinese hamster ovary (CHO) fibroblasts. (One, encoding apoB53, the N-terminal 53% of apoB100, can direct the assembly and secretion of lipoproteins when expressed in hepatoma cells, while the other, encoding the shorter apoB15, does not direct lipoprotein assembly.) Expression of apoB15 in CHO cells resulted in the accumulation of apoB15 protein in both medium and cells. In contrast, apoB was not detectable in medium or within CHO cells transfected with the plasmid encoding apoB53, despite the expression of apoB53 mRNA. ApoB53 did accumulate within transfected cells incubated with the thiol protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), suggesting that it is synthesized but completely degraded in the absence of the inhibitor. ApoB53 was not secreted despite its presence within ALLN-treated cells. Essentially all the apoB53 that accumulated in microsomes from ALLN-treated cells was associated with the membrane and was susceptible to degradation by exogenous trypsin, indicating exposure on the cytoplasmic face of the membrane. Thus, translocation of apoB53 across the endoplasmic reticulum membrane is blocked. However, the apoB53 bound to concanavalin A, suggesting that it is glycosylated and therefore partly exposed to the lumen as well. ApoB requires a unique process, not expressed in CHO fibroblasts, for its complete translocation and entrance into the secretory pathway. This process might account for the inability of abetalipoproteinemic patients to secrete apoB.
In vitro differentiation of embryonic stem cells into lymphocyte precursors able to generate T and B lymphocytes in vivo.
Authors: Gutierrez-Ramos J C , Palacios R
Published: May 2006

Embryonic stem cells can be induced in vitro, by coculture with the stromal line RP.0.10 and a mixture of interleukins 3, 6, and 7, to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+, PgP-1+, c-kit+, Joro75-, B-220-, F4/80-, Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny.
Activation of a D2 receptor increases electrical coupling between retinal horizontal cells by inhibiting dopamine release.
Authors: Harsanyi K , Mangel S C
Published: May 2006

In the fish retina, interplexiform cells release dopamine onto cone-driven horizontal cells. Dopamine decreases the electrical coupling between horizontal cells by activating adenylate cyclase through dopamine D1 receptors. Using intracellular recording, we have studied the effect of dopamine D2 receptor activation on horizontal cell electrical coupling in the intact goldfish retina. Superfusion of the D2 agonist LY171555 (quinpirole; 0.2-10 microM) increased horizontal cell coupling, as indicated by a decrease in responses to centered spots or slits of light. The length constant of the horizontal cell network increased an average of 31%. Although dopamine (0.5-20 microM) uncoupled horizontal cells, lower concentrations (e.g., 0.2 microM) initially uncoupled and then subsequently increased coupling beyond initial control levels. The coupling effect of LY171555 (10 microM) was blocked completely by prior application of the D1 agonist SKF 38393 at saturating (20 microM) or nonsaturating (2.5-5.0 microM) doses. Prior treatment of the retinas with 6-hydroxydopamine, which destroyed dopaminergic neurons, eliminated the coupling effect of LY171555 but not the uncoupling effect of SKF 38393. These results suggest that goldfish horizontal cells contain D1, but not D2, receptors and that dopamine activation of D2 autoreceptors on interplexiform cells inhibits dopamine release onto horizontal cells so that the electrical coupling between horizontal cells increases.
A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.
Authors: Schultheiss T , Choi J , Lin Z X , DiLullo C , Cohen-Gould L , Fischman D , Holtzer H
Published: May 2006

In many nonmuscle cells, nonsarcomeric alpha-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In striated muscle, a sarcomeric isoform of alpha-actinin (s-alpha-actinin) is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the alpha-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-alpha-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-alpha-actinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-alpha-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-like bodies. With time the affected myofibrils fragmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-alpha-actinin was significantly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "over-expressed" full-length or truncated protein did not self-aggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous alpha-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or post-translational modifications of s-alpha-actinin.
Regulation of assimilatory nitrate reductase activity in soil by microbial assimilation of ammonium.
Authors: McCarty G W , Bremner J M
Published: May 2006

It is well established that assimilatory nitrate reductase (ANR) activity in soil is inhibited by ammonium (NH4+). To elucidate the mechanism of this inhibition, we studied the effect of L-methionine sulfoximine (MSX), an inhibitor of NH4+ assimilation by microorganisms, on assimilatory reduction of nitrate (NO3-) in aerated soil slurries treated with NH4+. We found that NH4+ strongly inhibited ANR activity in these slurries and that MSX eliminated this inhibition. We also found that MSX induced dissimilatory reduction of NO3- to NH4+ in soil and that the NH4+ thus formed had no effect on the rate of NO-3 reduction. We concluded from these observations that the inhibition of ANR activity by NH4+ is due not to NH4+ per se but to products formed by microbial assimilation of NH4+. This conclusion was supported by a study of the effects of early products of NH4+ assimilation (L amino acids) on ANR activity in soil, because this study showed that the biologically active, L isomers of glutamine and asparagine strongly inhibited ANR activity, whereas the D isomers of these amino acids had little effect on ANR activity. Evidence that ANR activity is regulated by the glutamine formed by NH4+ assimilation was provided by studies showing that inhibitors of glutamine metabolism (azaserine, albizziin, and aminooxyacetate) inhibited ANR activity in soil treated with NO3- but did not do so in the presence of MSX.
Cloning and characterization of a mouse cDNA encoding a cytoplasmic protein-tyrosine-phosphatase.
Authors: Mosinger B , Tillmann U , Westphal H , Tremblay M L
Published: May 2006

A mouse cDNA encoding a non-receptor-type phosphotyrosine phosphatase (PTP; EC 3.1.3.48) has been isolated. The 1570-base-pair cDNA contains a single open reading frame that predicts a 382-amino acid protein with Mr 44,640. The nucleic acid and amino acid sequences are homologous to those of a previously described human T-cell PTP [Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., Fischer, E. H. & Krebs, E. G. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261]; however, the mouse and human 3' sequences diverge and predict markedly different protein carboxyl termini. The mouse PTP gene is expressed as a 1.9-kilobase message in several stages of murine embryonic development and in a variety of adult tissues. An additional 1.3-kilobase message was found to be expressed specifically in testes. Finally, we report the isolation of a human T-cell PTP cDNA containing a 3' end sequence homologous to the mouse PTP.
Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene.
Authors: Friling R S , Bergelson S , Daniel V
Published: May 2006

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, beta-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility. Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.
Testis-/embryo-expressed genes are clustered in the mouse H-2K region.
Authors: Yeom Y I , Abe K , Bennett D , Artzt K
Published: May 2006

The major histocompatibility complex (MHC) of the mouse is located on chromosome 17 in the distal inversion of the t complex. In addition to genes playing major roles in the immune response, it contains a diversity of genes. In humans, numerous diseases are known to be associated with the MHC loci. Moreover, at least three recessive embryonic t-lethal mutations have been mapped to the MHC. Here a molecular genetic approach was used to study the detailed genomic structure of 240 kilobases (kb) surrounding the H-2K gene and 150 kb of a partly homologous region located in the distal inversion of the t complex. Combined with previous findings, the H-2K region was found to contain an impressively high density of genes--12 transcription units in 240 kb. Surprisingly, virtually all of these genes are expressed in testis and/or embryos. The genomic organization of this region is contrasted with the 150 kb of the homologous area where only three genes and an endogenous retrovirus reside.
Nitric oxide stimulates auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.
Authors: Zhang J , Snyder S H
Published: May 2006

Nitric oxide generation in brain cytosolic fractions markedly enhances ADP-ribosylation of a single 37-kDa protein. By utilizing a biotinylated NAD and avidin affinity chromatography, we purified this protein. Partial amino acid sequencing establishes its identity as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This is further confirmed by detection of GAPDH enzymatic activity in the purified 37-kDa protein. GAPDH is ADP-ribosylated in the absence of brain extract. This auto-ADP-ribosylation is enhanced by nitric oxide generation. ADP-ribosylation appears to involve the cysteine where NAD interacts with GAPDH so that ADP-ribosylation likely inhibits enzymatic activity. Such inhibition may play a role in nitric oxide-mediated neurotoxicity.
Regulation of recombinant rat tyrosine hydroxylase by dopamine.
Authors: Ribeiro P , Wang Y , Citron B A , Kaufman S
Published: May 2006

Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.
Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes.
Authors: Sadoshima J , Takahashi T , Jahn L , Izumo S
Published: May 2006

Mechanical loading of cardiac and skeletal muscles in vivo and in vitro causes rapid activation of a number of immediate-early (IE) genes and hypertrophy of muscle cells. However, little is known as to how muscle cells sense mechanical load and transduce it into intracellular signals of gene regulation. We examined roles of putative cellular mechanotransducers, mechanosensitive ion channels, the cytoskeleton, and contractile activity in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channel that was completely blocked by gadolinium (Gd3+). Inhibition of this channel by Gd3+ did not affect either the stretch-induced expression of IE genes or the increase in protein synthesis. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression and increase in protein synthesis. Arresting contractile activity of myocytes by high K+, tetrodotoxin, or Ba2+ did not affect the stretch-induced IE gene expression. Tetrodotoxin-arrested myocytes could increase protein synthesis in response to stretch. These results suggest that Gd(3+)-sensitive ion channels, microtubules, microfilaments, and contractile activity may not be necessary for transduction of mechanical stretch into the IE gene expression and hypertrophy. The stimulus of membrane stretch may be transmitted to the cell nucleus through some mechanisms other than electrical or direct mechanical transduction in cardiac myocytes.
RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis.
Authors: Shepard A , Shiba K , Schimmel P
Published: May 2006

The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases. Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent. In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure. Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure. A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes. We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins. Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid. Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure. The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.
Cloning, genetic mapping, and expression analysis of an Arabidopsis thaliana gene that encodes 1-aminocyclopropane-1-carboxylate synthase.
Authors: Van der Straeten D , Rodrigues-Pousada R A , Villarroel R , Hanley S , Goodman H M , Van Montagu M
Published: May 2006

A genomic clone of one member of the Arabidopsis thaliana (L.) Heynh. 1-aminocyclopropane-1-carboxylate (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) gene family (AT-ACC1) was isolated and sequenced. A region of homology was found in the 5'-untranslated region with the promoter of a zucchini and a tomato ACC synthase gene. Comparison of its primary structure with other ACC synthases revealed conservation of seven peptide regions as well as similarity with 11 amino acids of the catalytic site of aminotransferases. Genomic DNA gel blotting suggested the existence of an ACC synthase multigene family in Arabidopsis, possibly with three other members, none of which is very closely related to AT-ACC1. The existence of at least one other gene was confirmed by the isolation of a cDNA (AT-ACC2) from a flower-specific cDNA library. The AT-ACC1 gene was mapped on the Arabidopsis restriction fragment length polymorphism map and is located on the top of chromosome 1. This position does not correspond to any known mutation on the genetic map. Expression of the AT-ACC1 gene was studied by reverse transcription-PCR on total RNA. Messenger accumulation was strong in young leaves and flowers. The gene was not induced by wounding of young leaves or in seedlings in the presence of auxin. Ethylene exposure of mature plants led to an induction of AT-ACC1 gene expression. It is suggested that AT-ACC1 protein has a role in developmental control of ethylene synthesis.
Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity.
Authors: Barbas C F , Crowe J E , Cababa D , Jones T M , Zebedee S L , Murphy B R , Chanock R M , Burton D R
Published: May 2006

Respiratory syncytial virus (RSV) is the most important cause, throughout the world, of severe viral lower respiratory tract illness in young children. Antibodies are known to mediate resistance to RSV infection and illness. We have isolated a number of human monoclonal Fab fragments to RSV F glycoprotein from a combinatorial antibody library expressed on the surface of phage. One of these neutralized a wide range of virus isolates, 10 subgroup A and 9 subgroup B isolates, with a titer (60% neutralization) of approximately 0.1-1.0 micrograms/ml. Another Fab neutralized diverse isolates at a concentration somewhat higher. These human Fab fragments show great promise for use in the prophylaxis or therapy of serious RSV lower respiratory tract disease. For intramuscular or intravenous administration, whole antibodies will be required, whereas for aerosol application, F(ab')2 or Fab fragments may suffice.
Acid-induced structural changes in human rhinovirus 14: possible role in uncoating.
Authors: Giranda V L , Heinz B A , Oliveira M A , Minor I , Kim K H , Kolatkar P R , Rossmann M G , Rueckert R R
Published: May 2006

X-ray diffraction data were collected from human rhinovirus 14 crystals a few minutes after exposure to acid vapor and prior to excessive crystalline disorder. Conformational changes occurred (i) in the GH loop of viral protein (VP) 1, (ii) at the ion binding site on the outer surface of the pentamer center, and (iii) in VP3 and VP4 on the virion's interior in the vicinity of the fivefold axis. Amino acid substitutions in mutants resistant to low pH, or to drugs that inhibit uncoating, were concentrated in the vicinity of the GH loop. It is proposed that the acid-induced changes reflect processes that trigger uncoating.
Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein.
Authors: Zhu D , Kidder G M , Caveney S , Naus C C
Published: May 2006

To examine the role of gap-junctional intercellular communication in controlling cell proliferation, we have transfected C6 glioma cells with connexin 43 cDNA. The growth of transfected clones was dramatically reduced compared with nontransfected glioma cells. To further characterize the role of gap junctions in controlling proliferation, we have examined the growth of C6 cells cocultured with transfected cells overexpressing connexin 43. Although C6 cells grew at their normal rate when cocultured with nontransfected C6 cells, when cocultured with connexin 43-overexpressing cells they displayed a dramatic reduction in growth rate. Furthermore, a significant, dose-dependent reduction in cell proliferation was noted when C6 cells were cultured in medium conditioned by transfected cells. This effect correlated with the level of connexin 43 expression. These results suggest that the decreased cell proliferation rate of transfected cells and C6 cells cultured with them is due to the secretion of a growth inhibitory factor(s) and that the secretion of this factor may be linked to the level of gap junctional intercellular communication.
Regulation of neurosecretory habituation in PC12 cells: parallel pathways used by cAMP and calcium.
Authors: Martin P T , Koshland D E
Published: May 2006

Habituation of norepinephrine secretion in PC12 cells serves as a model for understanding the molecular mechanisms of simple memory processes in neurons. Elevation of intracellular cAMP levels by incubation with N-ethylcarboxamidoadenosine (NECA) or forskolin increased norepinephrine secretion in response to depolarization by high potassium or stimulation with acetylcholine. The extent to which cAMP altered norepinephrine secretion was dependent on the timing of its elevation, and it also altered the rate of habituation under certain conditions. However, cAMP increased norepinephrine secretion by a pathway distinct from that governing internal calcium levels, which correlates with habituation in the absence of elevated cAMP. An inverse correlation was found between the ability of calcium to lower NECA-induced cAMP levels and the ability of calcium to cause norepinephrine secretion. A model is proposed in which a single calcium-dependent pathway modulates both norepinephrine secretion and cAMP metabolism.
Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor.
Authors: Ross T K , Prahl J M , Herzberg I M , DeLuca H F
Published: May 2006

The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.
Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1.
Authors: Perides G , Safran R M , Rueger D C , Charness M E
Published: May 2006

The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration.
Alternative splicing generates metabotropic glutamate receptors inducing different patterns of calcium release in Xenopus oocytes.
Authors: Pin J P , Waeber C , Prezeau L , Bockaert J , Heinemann S F
Published: May 2006

A splice variant of the metabotropic glutamate receptor (mGluR) 1a, named mGluR1c, was isolated. Compared to mGluR1a, the predicted mGluR1c protein is 302 amino acids shorter at its C-terminal end. Despite this difference, mGluR1c activates phospholipase C in Xenopus oocytes with a pharmacological profile identical to that of mGluR1a. However, in contrast to the large fast transient responses induced by mGluR1a, mGluR1c receptors elicit a small more slowly generated long-lasting oscillatory current, suggesting that these two receptors do not generate the same pattern of Ca2+ release in Xenopus oocytes. In situ hybridization data show that mGluR1c mRNA is expressed at a lower level than the other splice variants of mGluR1. Some differences in the regional distribution of these transcripts were observed in the cerebellum, the olfactory bulb, and the striatum.
Depth, motion, and static-flow perception at metaisoluminant color contrast.
Authors: Kovaćs I , Julesz B
Published: May 2006

Many experiments concerned with the role of color in depth and motion perception have applied isoluminant random-dot stereograms and cinematograms. The poor performance in the absence of luminance contrast has been associated with color-blindness of stereopsis and motion perception (Livingstone, M.S. & Hubel, D.H. (1987) J. Neurosci. 7, 3416-3468). Nevertheless, isoluminant stimuli are not fully accepted as appropriate tools in isolating central mechanisms (Logothetis, N.K., Schiller, P.H., Charles, E.R. & Hurlbert, A.C. (1990) Science 247, 214-217). In our experiments we use a broad luminance range to test whether color can contribute to a given mechanism when luminance contrast is present but has a strong "veto" effect from opposite luminance contrast, a condition we named "metaisoluminance." There is no fusion in stereopsis under polarity reversal, when only luminance information is given, and reversed-phi phenomenon is experienced for motion. As a third "matching" task, we included polarity-reversed random-dot Glass-patterns, which exhibit "static flow" and also show pattern reversal. We found that color can counteract the effects of polarity reversal by restoring stereoscopic fusion and reversed phi motion and does it with increased efficiency as the hue contrast increases. We found no such effect of color in Glass-patterns. Thus, we showed that the visual system for binocular depth and motion perception is not color-blind, although correlated hue information under metaisoluminance does not appear to yield shape perception.
Cloning and sequencing of a trophoblast-endothelial-activated lymphocyte surface protein: cDNA sequence and genomic structure.
Authors: Voland J R , Wyzykowski R J , Huang M , Dutton R W
Published: May 2006

We have previously described the distribution of a surface glycoprotein recognized by monoclonal antibody B721. We now report the molecular characterization of this molecule at the protein, cDNA, and genomic level. A 75-kDa glycoprotein can be immunoprecipitated from B721+ tissues. We have isolated a near full-length cDNA containing the complete coding region and a full-length genomic clone. We present evidence that this protein has similarity to several classes of nuclear transcription factors, particularly the myc family of proteins. The 721P protein was found to have a leucine zipper-like structure, a possible basic region immediately upstream from the leucine zipper, and a protein kinase A phosphorylation site. 721P protein is encoded by a gene distinct from any deposited in existing data bases, and displays several features associated with proteins involved in signal transduction and gene regulation.
Shared HLA class II-associated genetic susceptibility and resistance, related to the HLA-DQB1 gene, in IgA deficiency and common variable immunodeficiency.
Authors: Olerup O , Smith C I , Björkander J , Hammarström L
Published: May 2006

Most cases of selective IgA deficiency (IgA-D) and common variable immunodeficiency (CVID) occur sporadically. However, familial clustering is not uncommon, and the two disorders can occur within the same family. We have previously described positive associations with three DR-DQ haplotypes as well as a strong negative association with DRw15,DQw6,Dw2 in IgA-D. Different amino acids at position 57 of the HLA-DQ beta chain were found to be related to susceptibility and resistance to IgA-D. Now we have found identical, although somewhat weaker, positive and negative DR-DQ associations in a large group of CVID patients (n = 86), as well as the same associations with codon 57 of the DQB1 gene. In addition, we have confirmed our earlier observations in an independent group of IgA-D individuals (n = 69), and in sib-pair analysis we have found linkage of the genetic susceptibility to IgA-D to the HLA class II region. In IgA-D individuals not carrying the three overrepresented DR-DQ haplotypes, the same positive association with a non-aspartic acid residue at position 57 of the HLA-DQ beta chain was seen. The previously reported associations with deletions of the HLA class III genes C4A (fourth component of complement) and CYP21P (steroid 21-hydroxylase pseudogene) were, in our groups of immunodeficient individuals, statistically secondary to the association with the DQB1 allele 0201. The shared HLA class II associations in the two humoral immunodeficiencies support the hypothesis that IgA-D and CVID are related disorders. Disease susceptibility and resistance are most closely associated with a gene(s) within the DR-DQ region, alleles of the DQB1 locus being candidate genes.
DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase.
Authors: Mann S K , Yonemoto W M , Taylor S S , Firtel R A
Published: May 2006

We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.
Role of fibrinogen alpha and gamma chain sites in platelet aggregation.
Authors: Farrell D H , Thiagarajan P , Chung D W , Davie E W
Published: May 2006

Fibrinogen (Fbg) mediates platelet aggregation by its interaction with the platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3). Peptides containing the amino acid sequence RGD derived from the alpha chain (residues alpha 95-97 and residues alpha 572-574) and the sequence HHLGGAKQAGDV derived from the carboxyl terminus of the gamma chain of Fbg (residues gamma 400-411) inhibit these interactions. To determine the role of these sequences in intact Fbg, recombinant human Fbg (rFbg), mutant rFbgs with an RGD-->RGE substitution at either position alpha 97 or alpha 574, and a rFbg gamma'-containing variant that has a carboxyl-terminal interruption in the HHLGGAKQAGDV sequence have been expressed in transfected BHK cells. Purified rFbg and the two RGE mutant Fbgs were similar to plasma Fbg in platelet aggregation assays. In contrast, the gamma' variant Fbg was markedly defective in platelet aggregation. These data support the proposals that the carboxyl-terminal region of the gamma chain of Fbg is essential for optimal platelet aggregation and that the alpha-chain RGD sequences are neither necessary nor sufficient for platelet aggregation.
Saving the "library of life".
Authors: Benford G
Published: May 2006

A broad program of freezing species in threatened ecospheres could preserve biodiversity for eventual use by future generations. Sampling without studying can lower costs dramatically. Local labor can do most of the gathering. Plausible costs of collecting and cryogenically suspending the tropical rain forest species, at a sampling fraction of 10(-6), are about 2 billion dollars for a full century. Much more information than species DNA will be saved, allowing future biotechnology to derive high information content and perhaps even resurrect then-extinct species. Parallel programs of in situ and other ex situ preservation are essential to allow later expression of frozen genomes in members of the same genus. This is a broad proposal that should be debated throughout the entire scientific community.