bluuebunny/crossref_metadata_2025 huggingface dataset:
Free energy of sickling: A simulation analysis.
Authors: Kuczera K , Gao J , Tidor B , Karplus M
Published: May 2006

Molecular dynamics simulations were performed to calculate the difference between the dimerization free energies of normal human deoxyhemoglobin (HbA) and the mutant sickle-cell deoxyhemoglobin HbS (Glu-beta 6----Val) for one of the lateral contacts in the HbS x-ray structure. The simulations yield a value of--15 kcal/mol. Although there is no quantitative experimental value for comparison, this is in qualitative agreement with the experimental result that HbS self-assembles into multistranded fibers that are responsible for erythrocyte sickling, while HbA does not. The free-energy difference was decomposed into enthalpic and entropic terms, both of which are significant, and the contributions of individual protein residues and of the solvent were examined. Electrostatic effects play the dominant role in favoring dimerization of HbS compared with HbA; van der Waals interactions make a negligible contribution to the difference. Both differential solvation and protein-protein interactions are important. Interactions within the donor tetramer (i.e., that containing the Glu-beta 6 mutation site), as well as those with the acceptor tetramer, contribute to the preferential free energy of dimerization of HbS.
Volume-sensitive K-Cl cotransport in inside-out vesicles made from erythrocyte membranes from sheep of low-K phenotype.
Authors: Kracke G R , Dunham P B
Published: May 2006

Unidirectional K ion effluxes were measured from inside-out vesicles prepared from erythrocyte membranes from sheep of the low-K phenotype. Total K efflux was 150 nmol per mg of protein per hr in a Cl medium of 295 mosmol/kg (with the Na/K pump inhibited). Cl-dependent K efflux (determined with methanesulfonate replacing Cl) was 54 nmol/(mg.hr). Cl-dependent K efflux (K-Cl cotransport) increased to 77 nmol/(mg.hr) with osmotic swelling of approximately 30% in 230-mosmol/kg medium and decreased to 13 nmol/(mg.hr) after shrinkage of approximately 60% in 430-mosmol/kg medium. Osmotically induced changes in transport and vesicle volume were reversible. K-Cl cotransport was enhanced by ATP. Nonhydrolyzable ATP analogues failed to substitute for ATP, indicating that phosphorylation is involved. However, in the absence of added ATP there was significant K-Cl cotransport, suggesting that phosphorylation is not essential for function. The results provide clues about the nature of the signals detected by the sensor of cell volume changes and demonstrate that inside-out vesicles from sheep erythrocyte membranes provide an advantageous experimental system for investigation of the volume sensor.
Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene.
Authors: Itaya M
Published: May 2006

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.
Differentiation of F9 embryonal carcinoma cells induced by the c-jun and activated c-Ha-ras oncogenes.
Authors: Yamaguchi-Iwai Y , Satake M , Murakami Y , Sakai M , Muramatsu M , Ito Y
Published: May 2006

The activated c-Ha-ras oncogene induced AP1-site DNA-binding activity in F9 cells. This induction appeared to be due, at least in part, to the induction of c-jun transcription. Both activated c-Ha-ras and c-jun induced the differentiation of F9 cells to endoderm-like cells. Thus, AP1 appears to play a key role in the initial stage of F9 cell differentiation.
Effects on adaptedness of variations in ribosomal DNA copy number in populations of wild barley (Hordeum vulgare ssp. spontaneum).
Authors: Zhang Q F , Saghai Maroof M A , Allard R W
Published: May 2006

Twenty alleles, 12 at Mendelian locus Rrn1 and 8 at locus Rrn2, control rRNA genes [ribosomal DNA (rDNA)] variability in barley. These alleles differ strikingly in their effects on adaptedness. In the present study, we determined variation in the copy number of 101 accessions of wild barley plants from 10 ecologically diverse sites in Israel and examined relationships between rDNA copy number and adaptedness. The average multiplicity of rDNA per haploid genome was 1881 copies and the average numbers of copies for Rrn1 and Rrn2 were 962 and 917, respectively. The total number of copies as well as the number of copies for Rrn1 and Rrn2 varied widely from plant to plant within sites and also from site to site. The predominant allele of Rrn2 had somewhat more copies on the average than the other alleles of this locus but differences between the predominant allele and other alleles of Rrn1 were not statistically significant. Overall, the results indicated that differing amounts of rDNA resulting from variations in copy number and/or number of subrepeats in the intergenic spacer region were not closely associated with adaptedness. This suggests that the high adaptedness of a few specific alleles results in large part from adaptatively favorable nucleotide sequences in the transcription units and/or the intergenic spacer regions of the favored alleles--i.e., that adaptedness in barley depends on the quality more than on the quantity of rDNA present.
X-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor.
Authors: Swain A L , Miller M M , Green J , Rich D H , Schneider J , Kent S B , Wlodawer A
Published: May 2006

The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-psi[CH(OH)CH2N]-Pro-Ile-Val-OMe; the Ki is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.
Molecular cloning of complementary DNAs encoding two cationic peroxidases from cultivated peanut cells.
Authors: Buffard D , Breda C , van Huystee R B , Asemota O , Pierre M , Ha D B , Esnault R
Published: May 2006

We have isolated, cloned, and characterized two cDNAs corresponding to the mRNAs for cationic peroxidases synthesized by cultured peanut cells. The first clone was obtained from a phage lambda gt11 library screened with antibodies directed against the major secreted isozyme. Its predicted amino acid sequence, deduced from the 1228-base-pair (bp) cDNA, revealed a 22-amino acid signal peptide and a 294-amino acid mature protein (Mr, 31,228). The second clone was isolated from a lambda gt10 library screened with oligonucleotides corresponding to the regions for acid/base catalysis and the fifth ligand of heme. This cDNA (1344 bp) encodes a protein (330 amino acids) with a mature peptide of 307 residues (Mr, 32,954). The two peanut peroxidases are 46% homologous. The estimated gene copy numbers of these peroxidases might be close to 1 or 2 per haploid genome. A comparison of the amino acid sequence of these peanut peroxidases with other known isozymes shows two already known regions of homology (the region for acid/base catalysis and the fifth ligand of heme). Moreover, some new characteristics appeared such as a glycosylation site identical in five of the seven isozymes, a putative antigenic determinant common to all the isozymes, and a region of the highest homology. A secondary structure prediction showed that it corresponds to a 16-amino acid helix linked to the next one by a long stretch of beta strands and coils and might represent a critical structural element.
A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni.
Authors: Rajkovic A , Davis R E , Simonsen J N , Rottman F M
Published: May 2006

We present evidence that a subset of mRNAs in the human parasitic trematode Schistosoma mansoni contain an identical 36-nucleotide spliced leader (SL) sequence at their 5' termini. The SL is derived from a 90-nucleotide nonpolyadenylylated RNA (SL RNA), presumably by trans-splicing. Neither the SL nor the SL RNA share significant sequence identity with previously described trans-spliced leaders and SL RNAs in trypanosomatid protozoans or nematodes. However, several features, such as predicted secondary structure, trimethylguanosine cap, and potential Sm binding site, suggest similarities among SL RNAs in widely divergent organisms. Our evidence also indicates that the exon 3 acceptor site of the 3-hydroxy-3-methylglutaryl-CoA reductase gene can be spliced either to the SL by trans-splicing or to an upstream exon, 2, by cis-splicing. The presence of a SL sequence in S. mansoni, a member of the phylum Platyhelminthes, suggests that transplicing may be a common feature of other lower invertebrates.
Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein.
Authors: Chambers T J , Weir R C , Grakoui A , McCourt D W , Bazan J F , Fletterick R J , Rice C M
Published: May 2006

Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cis-acting site-specific proteolysis at the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis of the His-53, Asp-77, and Ser-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.
Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.
Authors: Rogers K V , Goldman P S , Frizzell R A , McKnight G S
Published: May 2006

Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.
Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.
Authors: Garcia-Morales P , Minami Y , Luong E , Klausner R D , Samelson L E
Published: May 2006

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this reagent to investigate the effect of tyrosine phosphatase inhibition in a murine T-cell hybridoma. Increasing concentrations of phenylarsine oxide result in an increase in tyrosine phosphate on a number of intracellular substrates in unstimulated T cells, suggesting that a protein-tyrosine kinase is constitutively active in these cells. The effect of phenylarsine oxide on T cells stimulated with an anti-Thy 1 monoclonal antibody is more complex. At low concentrations of drug, there is a synergistic increase in the level of tyrosine phosphate on certain cellular substrates. At higher concentrations, anti-Thy 1-stimulated tyrosine phosphorylation is inhibited. These results indicate that tyrosine phosphorylation in T cells is tightly regulated by tyrosine phosphatases. Partial inhibition of these enzymes results in enhanced substrate phosphorylation. Inhibition of all stimulated tyrosine phosphorylation by high doses of phenylarsine oxide suggests that tyrosine kinase activity is regulated by tyrosine phosphatases.
Multiple stores of calcium are released in the sea urchin egg during fertilization.
Authors: Rakow T L , Shen S S
Published: May 2006

Fertilization initiates a transient increase in intracellular Ca2+ principally by Ca2+ release from intracellular stores. Possible multiple Ca2+ stores and multiple receptor regulation of the same store have been reported. Here we report the presence of at least two independent intracellular Ca2+ stores in the sea urchin egg, which are released during fertilization. Ca2+ release from one store is mediated by inositol 1,4,5-trisphosphate (IP3) and is sensitive to low molecular weight heparin. The other store is heparin insensitive and independent of IP3 regulation, but the regulatory factor remains unidentified. A transient increase in Ca2+ in heparin-loaded eggs is observed during fertilization, which suggests that IP3-independent Ca2+ release mediates the production of IP3 and release of the IP3-dependent store. Experiments presented here do not support the idea of sperm receptor coupling to inositol phospholipid hydrolysis through a GTP-binding protein mediating the fertilization response.
Regulation of interleukin 2-dependent growth responses by glycosylphosphatidylinositol molecules.
Authors: Merida I , Pratt J C , Gaulton G N
Published: May 2006

The molecular mechanism of signal transduction through the interleukin 2 (IL-2) receptor remains an enigma. Glycosylphosphatidylinositol (GPI) lipids were investigated as one component of this process. IL-2 stimulated the rapid (30 sec) loss of greater than 50% of GPI in the IL-2-dependent T-cell line CTLL-2. Half-maximal GPI loss was detected at 40 pM IL-2, coincident with the EC50 (20 pM) for IL-2-induced proliferation of this cell line. This effect was specifically inhibited by antibodies that bind either IL-2 or the IL-2 receptor. The loss of GPI was mirrored by the accumulation of both polar inositolphosphoglycan (IPG) and diacylglycerol lipid fragments within cells. Increases in lipids were initially restricted to myristoyl diacylglycerol but were followed by the accumulation of myristoyl phosphatidic acid. These results are indicative of IL-2-dependent hydrolysis of GPI in T cells. The biological relevance of this hydrolysis was demonstrated by synergism of purified IPG with IL-2 in T-cell proliferation responses. The inclusion of IPG (0.1 microM) in determinations of IL-2-dependent CTLL-2 growth shifted the EC50 from 20 to 7 pM IL-2. IPG had no effect on either the number or affinity of IL-2 receptors; therefore, half-maximal CTLL-2 proliferation was obtained at less than 10% IL-2 receptor occupancy. These results demonstrate that GPI lipids are an important component of the biological response to IL-2.
Lipophilic halogenated congeners of 2',3'-dideoxypurine nucleosides active against human immunodeficiency virus in vitro.
Authors: Shirasaka T , Murakami K , Ford H , Kelley J A , Yoshioka H , Kojima E , Aoki S , Broder S , Mitsuya H
Published: May 2006

Four 2-amino-6-halo- and four 6-halo-2',3'-dideoxypurine ribofuranosides (ddPs) were synthesized and tested for in vitro activity to suppress the infectivity, cytopathic effect, Gag protein expression, and DNA synthesis of human immunodeficiency virus (HIV). The comparative order of in vitro anti-HIV activity of the eight 6-halo-ddPs was as follows: 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro greater than 2-amino-6-bromo greater than 2-amino-6-iodo, 6-chloro greater than 6-bromo greater than 6-iodo. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddPs showed a potent activity against HIV comparable to that of 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG) and completely blocked the infectivity of HIV without affecting the growth of target cells. The lipophilicity order was as follows: 2-amino-6-iodo greater than 2-amino-6-bromo greater than 2-amino-6-chloro greater than 2-amino-6-fluoro much greater than ddG greater than ddI. All eight 6-halo-ddPs were substrates for adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4). The relative rates of hydrolysis by ADA were as follows: ddA, 2-amino-6-fluoro much greater than 2-amino-6-chloro, 2-amino-6-bromo greater than 2-amino-6-iodo. Taken together, these compounds may represent an additional class of lipophilic prodrugs for ddI and ddG and may also provide a strategy for endowing therapeutic purine nucleosides with desirable lipophilicity.
Primary combined immunodeficiency resulting from defective transcription of multiple T-cell lymphokine genes.
Authors: Chatila T , Castigli E , Pahwa R , Pahwa S , Chirmule N , Oyaizu N , Good R A , Geha R S
Published: May 2006

The circulating T lymphocytes of a female child with recurrent opportunistic infections were normal in number and phenotype but exhibited poor proliferation and decreased synthesis of the T-cell growth factor interleukin (IL) 2 in response to mitogens. Recombinant IL-2 fully restored the proliferative responses of her T cells, suggesting that her poor immune function was related to IL-2 deficiency. Northern blot analysis of total cellular RNA from the patient's T cells revealed markedly decreased levels of IL-2 mRNA of normal size. In addition, mRNA levels of other lymphokines selectively expressed by T cells, which include IL-3, IL-4, and IL-5, were either severely depressed or absent. The levels of interferon gamma mRNA were moderately decreased, while those of granulocyte-macrophage colony stimulating factor, a lymphokine the production of which is not restricted to T cells, were unaffected. The decreased level of lymphokine mRNA in the patient's T lymphocytes was not from enhanced catabolism but resulted from a diminution in the transcription rate of the affected lymphokine genes. Normal transduction via the T-cell receptor/CD3 complex of biochemical signals necessary for the initiation of lymphokine gene transcription indicated that the defect was distal to the membrane signal-transducing apparatus. The defect is hypothesized to involve a T-cell-specific trans-acting regulatory factor required for transcription of the affected lymphokine genes.
Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5'-[beta,gamma-imido]triphosphate.
Authors: Schnapp B J , Crise B , Sheetz M P , Reese T S , Khan S
Published: May 2006

Kinesin is a microtubule-activated ATPase that moves objects toward the plus end of microtubules and makes microtubules glide along a glass surface. Here we investigate a remarkable effect of the nonhydrolyzable analogue of ATP, adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA), on kinesin-driven microtubule gliding. Microtubule gliding that has been blocked by rapid replacement of ATP with p[NH]ppA requires 1-2 min of exposure to ATP before microtubule gliding resumes. This latency is not shortened by prolonged washing of p[NH]ppA-blocked microtubules in nucleotide-free buffer for up to 15 min, suggesting that ATP binding to a second nucleotide binding site on kinesin triggers the release of bound p[NH]ppA. To test this hypothesis, the release of [3H]p[NH]ppA from kinesin-microtubule complexes was followed in parallel biochemical assays. In nucleotide-free buffer, the bound p[NH]ppA was released over several hours from the complexes. However, addition of ATP caused the release of p[NH]ppA from the kinesin-microtubule complexes within 2 min, which was similar to the latent period for start-up of microtubule gliding after p[NH]ppA inhibition. The stoichiometry of p[NH]ppA bound per kinesin heavy chain at saturation was estimated to be approximately 1:2. These results suggest a model in which each molecule of kinesin has at least two nucleotide binding sites that alternately bind nucleotide.
The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.
Authors: Sweeney H L , Brito R M , Rosevear P R , Putkey J A
Published: May 2006

Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family.
Adenovirus early region 4 stimulates mRNA accumulation via 5' introns.
Authors: Nordqvist K , Akusjärvi G
Published: May 2006

The adenovirus major late transcription unit accounts for most virus-specific transcription late after infection. All mRNAs expressed from this unit carry a short spliced leader, the so-called tripartite leader, attached to their 5' ends. Here we describe a function for an adenovirus gene product in the control of major late mRNA abundance. We show that early region 4 (E4) stimulates mRNA accumulation from tripartite leader intron-containing transcription units approximately 10-fold in short-term transfection assays. The effect was already detectable in nuclear RNA and was not due to a transcriptional activation through any of the major late promoter elements or through an effect at nuclear to cytoplasmic mRNA transport. A surprising positional effect of the intron was noted. To be E4 responsive, the intron had to be placed close to the pre-mRNA 5' end. The same intron located far downstream in the 3' untranslated region of the mRNA was not E4 responsive. The E4 enhancement was not dependent on specific virus exon or intron sequences. These results suggest that E4 modulates a general pathway in mammalian mRNA formation.
Anti-Fas monoclonal antibody is cytocidal to human immunodeficiency virus-infected cells without augmenting viral replication.
Authors: Kobayashi N , Hamamoto Y , Yamamoto N , Ishii A , Yonehara M , Yonehara S
Published: May 2006

A cytotoxic monoclonal antibody (anti-Fas mAb) against the 200-kDa cell surface Fas antigen, which is associated with the tumor necrosis factor (TNF) receptor, was examined for its in vitro activity on human immunodeficiency virus (HIV)-infected cells. It was found that both TNF and anti-Fas mAb selectively killed the chronically HIV-infected cells. Uninfected cells were less sensitive to the antibody than those infected with HIV. When the cells were cultured in the presence of anti-Fas mAb immediately after the HIV infection, the spread of HIV-infected cells was suppressed by the antibody. TNF augmented both the synthesis of HIV-specific mRNA in HIV-infected cells and formation of multinucleated giant cells. In contrast, the anti-Fas mAb did not augment HIV replication or enhance the HIV-induced formation of syncytia. The results indicated that anti-Fas mAb mimicks the cytocidal action of TNF but does not augment HIV replication.
Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.
Authors: Gerwert K , Souvignier G , Hess B
Published: May 2006

Absorbance changes in the infrared and visible spectral range were measured in parallel during the photocycle of light-adapted bacteriorhodopsin, which is accompanied by a vectorial proton transfer. A global fit analysis yielded the same rate constants for the chromophore reactions, for protonation changes of protein side groups, and for the backbone motion. From this result we conclude that all reactions in various parts of the protein are synchronized to each other and that no independent cycles exist for different parts. The carbonyl vibration of Asp-85, indicating its protonation, appears with the same rate constant as the Schiff base deprotonation. The carbonyl vibration of Asp-96 disappears, indicating most likely its deprotonation, with the same rate constant as for the Schiff base reprotonation. This result supports the proposed mechanism in which the protonated Schiff base, a deprotonated aspartic acid (Asp-85) on the proton-release pathway, and a protonated aspartic acid (Asp-96) on the proton-uptake pathway act as internal catalytic proton-binding sites.
Specificity of cell-cell coupling in rat optic nerve astrocytes in vitro.
Authors: Sontheimer H , Minturn J E , Black J A , Waxman S G , Ransom B R
Published: May 2006

Intercellular coupling was studied in cultured rat optic nerve astrocytes individually characterized by A2B5 antibody staining. The presence of cell coupling was assessed by injecting single cells with the low molecular weight fluorescent dye Lucifer yellow and noting dye passage into adjacent cells; cell coupling was also studied by analyzing the decay phase of current transients recorded in response to small voltage steps using whole-cell patch-clamp recording. Cell coupling was restricted to A2B5- astrocytes, the majority of which had a flat fibroblast-like appearance and was never observed in A2B5+ stellate-shaped astrocytes. Furthermore, A2B5- astrocytes showed coupling only to A2B5- and never to A2B5+ astrocytes. Analysis of current transients provided an additional indicator for cell coupling. Astrocytes that showed dye coupling to at least one neighboring cell required the sum of two exponential functions to fit current transients, whereas a single exponential function sufficed to fit transients in cells that were not dye coupled. The specificity of cell coupling in cultured rat optic nerve astrocytes suggests that predominantly A2B5- astrocytes comprise a coupled glial syncytium; this physiological feature of these cells may be a specialized adaptation for "spatial buffering," the transport of K+ away from areas of focal extracellular accumulation. On the other hand, A2B5+ astrocytes form an uncoupled subpopulation of rat optic nerve glial cells that may serve different functions.
Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection.
Authors: Yagi T , Ikawa Y , Yoshida K , Shigetani Y , Takeda N , Mabuchi I , Yamamoto T , Aizawa S
Published: May 2006

In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half of the cells integrating with the neomycin phosphotransferase gene were homologous recombinants.
Frequent mutation of the p53 gene in human esophageal cancer.
Authors: Hollstein M C , Metcalf R A , Welsh J A , Montesano R , Harris C C
Published: May 2006

Sequence alterations in the p53 gene have been detected in human tumors of the brain, breast, lung, and colon, and it has been proposed that p53 mutations spanning a major portion of the coding region inactivate the tumor suppressor function of this gene. To our knowledge, neither transforming mutations in oncogenes nor mutations in tumor suppressor genes have been reported in human esophageal tumors. We examined four human esophageal carcinoma cell lines and 14 human esophageal squamous cell carcinomas by polymerase chain reaction amplification and direct sequencing for the presence of p53 mutations in exons 5, 6, 7, 8, and 9. Two cell lines and five of the tumor specimens contained a mutated allele (one frameshift and six missense mutations). All missense mutations detected occurred at G.C base pairs in codons at or adjacent to mutations previously reported in other cancers. The identification of aberrant p53 gene alleles in one-third of the tumors we tested suggests that mutations at this locus are common genetic events in the pathogenesis of squamous cell carcinomas of the esophagus.
Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin.
Authors: Hyde D R , Mecklenburg K L , Pollock J A , Vihtelic T S , Benzer S
Published: May 2006

From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner.
Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.
Authors: Tsurimoto T , Stillman B
Published: May 2006

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.
ADP-ribosylation factor is functionally and physically associated with the Golgi complex.
Authors: Stearns T , Willingham M C , Botstein D , Kahn R A
Published: May 2006

ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.
Tissue distribution of hepatopoietin-A: a heparin-binding polypeptide growth factor for hepatocytes.
Authors: Zarnegar R , Muga S , Rahija R , Michalopoulos G
Published: May 2006

Hepatopoietin-A (HPTA) is a heparin-binding polypeptide growth factor which consists of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively. It stimulates DNA synthesis in primary cultures of normal rat hepatocytes in serum-free medium. The complete purification and characterization of HPTA from rabbit serum were reported by us elsewhere. Recently we have determined the amino-terminal amino acid sequence of the rabbit HPTA light chain up to 24 residues and have shown that the sequence is not homologous with other known sequences. [N.B. Human hepatocyte growth factor, recently sequenced by two other groups, is the same molecular species as HPTA.] In the present paper we report the production of a neutralizing polyclonal antiserum raised in chicken against purified rabbit HPTA. This antiserum does not inhibit the mitogenic effect of other potent inducers of hepatocyte DNA synthesis (epidermal growth factor or acidic fibroblast growth factor), nor does it interact with these growth factors in an enzyme-linked immunosorbent assay (ELISA). The antibody recognizes HPTA, as was determined by Western immunoblotting. Since the tissue origin of HPTA is not known, this anti-HPTA antiserum was used to investigate the tissue distribution of HPTA in rabbits by immunohistostaining methods. Acinar cells of the pancreas, neurons of the brain, C cells of the thyroid, ductal cells of the salivary glands, and Brunners glands of the duodenum stained with anti-HPTA antibody. Liver, spleen, thymus, and kidney do not seem to contain appreciable amounts of HPTA. We confirmed these findings by extracting and purifying active HPTA from the stained tissues listed above. The anti-HPTA antibody recognizes HPTA purified from different tissues, as was determined by ELISA, Western immunoblotting, and immunoneutralization experiments.
Phage shock protein, a stress protein of Escherichia coli.
Authors: Brissette J L , Russel M , Weiner L , Model P
Published: May 2006

Filamentous phage infection induces the synthesis of large amounts of an Escherichia coli protein, phage shock protein (Psp), the product of a previously undescribed gene. This induction is due to the phage gene IV protein, pIV, an integral membrane protein. The uninduced level of Psp is undetectable, but when induced by prolonged synthesis of pIV, it can become one of the most abundant proteins in the cell. Psp is also synthesized transiently in response to several stresses (heat, ethanol, and osmotic shock). High-level synthesis occurs only after extreme treatment. Unlike the members of the heat shock regulon, Psp induction does not require the heat shock sigma factor, sigma 32; some stimuli that elicit sigma 32-dependent heat shock proteins do not induce Psp synthesis. The level of Psp induction after extreme stress is even higher in sigma 32 mutant cells, which are unable to mount a normal heat shock response, suggesting that these parallel stress responses are interrelated.
A strategy for fine-structure functional analysis of a 6- to 11-centimorgan region of mouse chromosome 7 by high-efficiency mutagenesis.
Authors: Rinchik E M , Carpenter D A , Selby P B
Published: May 2006

A refined functional map of a 6- to 11-centimorgan region surrounding the albino (c) locus in mouse chromosome 7 is being generated by N-ethyl-N-nitrosourea (EtNU) "saturation" mutagenesis of stem-cell spermatogonia. In the first phase of an experiment that will eventually test at least 3000 gametes, we screened 972 mutagenized gametes for the induction of both lethal and visible mutations with a two-cross breeding protocol. Thirteen mutations mapping within the limits of a segment corresponding to the cytologically visible Df(c Mod-2 sh-1)26DVT deletion were recovered. They represented three phenotypic groups: prenatal lethality (six mutations); a fitness/runting syndrome (three mutations, provisionally designated as fit variants); and a neurological/balance-defect abnormality (four mutations). Complementation analysis provided evidence for a true repeat mutation at the sh-1 (shaker-1) locus (for the neurological mutations) and another at the here defined fit-1 (fitness-1) locus. In addition, four complementation groups were defined by induced lethal mutations; the two other lethal mutations were each part of a cluster. The recovery of the repeat mutations suggests that the EtNU-induced mutation rate, estimated from specific-locus tests, should make it possible to achieve saturation mutagenesis of a chromosomal region. This experiment is providing basic logistical and statistical information on which to base strategies for expanding the functional map of larger segments of the mouse genome by experimental mutagenesis. It is also yielding additional mutations useful in dissecting the functional and molecular complexity of this segment of chromosome 7.
Pioneer neurons in the mouse trigeminal sensory system.
Authors: Stainier D Y , Gilbert W
Published: May 2006

Pioneer neurons establish preliminary nerve pathways that are followed by later-growing axons. The existence of pioneers and their importance is well documented in invertebrate systems. In mammals, early neuronal development has generally been difficult to study because of the size and complexity of the embryos, and the lack of adequate markers. Here we look at the time of earliest axonal outgrowth in the mouse embryo by using specific monoclonal antibodies to stain wholemount preparations. During the period of formation and closure of the neuropore beginning at embryonic day 8.5, we can follow the earliest trigeminal sensory neurons extending axons along stereotyped pathways. In the trigeminal ganglion, an early wave of neurogenesis gives rise to a small number of neurons whose axons pioneer the different trigeminal tracts in the periphery. After a brief pause (12 hr), these primary axons branch out to innervate individual targets. Emerging a day later, secondary fibers extend along the pioneers. By contrast, in the central nervous system, neurons of the mesencephalic trigeminal nucleus extend toward the rhombencephalon independently, ignoring preexisting fibers. These results show the existence of an early set of axonal tracts in the mouse peripheral nervous system that may be used for the guidance of later-differentiating neurons.
Primary structure of rat RT6.2, a nonglycosylated phosphatidylinositol-linked surface marker of postthymic T cells.
Authors: Koch F , Haag F , Kashan A , Thiele H G
Published: May 2006

RT6 is an unusual cell membrane protein that is expressed exclusively by postthymic T cells. The inherent defect in its expression has been correlated to lymphopenia and genetically determined susceptibility for insulin-dependent diabetes mellitus in the rat. We report here the primary structure of the RT6.2 alloantigen as deduced from the cDNA sequence. The predicted amino acid sequence of RT6.2 begins with a conventional leader of 20 amino acids and ends in a hydrophobic C-terminal extension peptide of 29 amino acids as is common for phosphatidylinositol-anchored proteins. Native RT6.2 is predicted to comprise 226 amino acids, with a calculated Mr of 26,036. Four cysteine residues account for two intrachain disulfide bonds. The sequence lacks potential N-glycosylation sites and contains an excess of positively charged residues. Homology searches in protein sequence data banks suggest that RT6.2 is not encoded by a member of the immunoglobulin supergene family. Moreover, these analyses did not reveal any close homologies of RT6.2 to known proteins: the highest homology found was 21.2% identity in a 52-amino acid overlap to the torpedo acetylcholinesterase precursor. Southern blot analyses indicate that RT6.2 is the product of a single-copy gene and provide evidence for closely related genes in the mouse and other species. The corresponding gene products remain to be identified.
Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis.
Authors: Miyamura T , Saito I , Katayama T , Kikuchi S , Tateda A , Houghton M , Choo Q L , Kuo G
Published: May 2006

A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatitis (NANBH). We have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. In contrast to the patients of chronic hepatitis, the antibody was barely detected in patients with a single episode of ALT elevation (1 out of 6). Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented. The antibody assay thus can be used for diagnosis and blood screening for PT-NANBH.
Subcellular distribution of small GTP binding proteins in pancreas: identification of small GTP binding proteins in the rough endoplasmic reticulum.
Authors: Nigam S K
Published: May 2006

Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[gamma-35S]) was assayed in each fraction. Enrichment of GTP[gamma-35S] binding was greatest in the interfacial "smooth" microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a [alpha-32P]GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP[gamma-35S] binding, as well as the small GTP binding proteins detected by the [alpha-32P]GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMs were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP[gamma-35S] binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.