bluuebunny/crossref_metadata_2025 huggingface dataset:
Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.
Authors: Børresen A L , Hovig E , Smith-Sørensen B , Malkin D , Lystad S , Andersen T I , Nesland J M , Isselbacher K J , Friend S H
Published: May 2006

At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostaining techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or p144D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.
Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa.
Authors: Ruf W , Edgington T S
Published: May 2006

Tissue factor (TF) binds the serine protease coagulation factor VIIa and initiates the coagulation protease cascade by forming a catalytic cofactor-enzyme complex. Using a photoactivatable crosslinking reagent coupled to factor VIIa, we have identified interactive sites in the amino-terminal (residues 44-84) and the carboxyl-terminal (residues 129-169) aspect of the extracellular domain of TF. Epitopes of inhibitory antibodies have previously indicated participation of these regions in TF function. The presence of the gamma-carboxyglutamic acid domain in factor VIIa appears to facilitate the interaction with the negatively charged, amino-proximate site, whereas crosslinking of TF with VIIa or des-(1-38)-VIIa at the positively charged carboxyl-proximate site was similar. Lack of alpha-helical secondary structure in the TF extracellular domain is consistent with the proposed structural similarity of TF with the cytokine receptor family. The interactive sites identified for TF are located in sequence spans that demonstrate a low degree of sequence conservation among the members of this receptor family. Regions with highly conserved residues, such as sequences encoded by exon 2 and 5 in TF, were not implicated in ligand recognition, suggesting that conserved residues in the receptor family may maintain the common beta-strand architecture, and variable regions provide a pair of nonidentical motifs for oriented ligand recognition.
Direct evidence for in vivo hydroxyl-radical generation in experimental iron overload: an ESR spin-trapping investigation.
Authors: Burkitt M J , Mason R P
Published: May 2006

Although the hydroxyl radical is often implicated as the species responsible for the initiation of oxidative damage in iron-overload conditions, no ESR evidence for the formation of the radical in vivo has been reported. We have employed a secondary radical-trapping technique in which the hydroxyl radical reacts with dimethyl sulfoxide to form the methyl radical, which is then detected as its adduct of the spin trap N-t-butyl-alpha-phenylnitrone in the bile of animals given an intragastric dose of ferrous sulfate. The identity of this adduct was verified by isotope-substitution techniques. We show that unless measures are taken to inactivate the iron excreted in the bile of treated animals, reactions between iron, oxygen, dimethyl sulfoxide, N-t-butyl-alpha-phenylnitrone, and bile components lead to the formation of artifacts during sample collection.
Vitamin D3 binding protein (group-specific component) is a precursor for the macrophage-activating signal factor from lysophosphatidylcholine-treated lymphocytes.
Authors: Yamamoto N , Homma S
Published: May 2006

A brief (30 min) treatment of mouse peritoneal cells (mixture of nonadherent lymphocytes and adherent macrophages) with 1-20 micrograms of lysophosphatidylcholine (lyso-PC) per ml in serum-supplemented RPMI medium 1640, followed by a 3-hr cultivation of the adherent cells alone, results in a greatly enhanced Fc receptor-mediated phagocytic activity of macrophages. This rapid process of macrophage activation was found to require a serum factor, the vitamin D3 binding protein (the human protein is known as group-specific component; Gc). Efficient activation of macrophages was achieved by using medium containing purified human Gc protein. Analysis of intercellular signal transmission among nonadherent (B and T) cells revealed that lyso-PC-treated B cells modify Gc protein to yield a proactivating factor, which can be converted by T cells to the macrophage-activating factor. This rapid generation process of the macrophage-activating factor was also demonstrated by stepwise incubation of Gc protein with lyso-PC-treated B-cell ghosts and untreated T-cell ghosts, suggesting that Gc protein is modified by preexisting membranous enzymes to yield the macrophage-activating factor. Incubation of Gc protein with a mixture of beta-galactosidase and sialidase efficiently generated the macrophage-activating factor. Stepwise incubation of Gc protein with B- or T-cell ghosts and sialidase or beta-galactosidase revealed that Gc protein is modified by beta-galactosidase of B cells and sialidase of T cells to yield the macrophage-activating factor. Administration to mice of a minute amount (4-10 pg per mouse) of in vitro, enzymatically generated macrophage-activating factor resulted in a greatly enhanced (3- to 7-fold) ingestion activity of macrophages.
Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA.
Authors: Alderson A , Sabelli P A , Dickinson J R , Cole D , Richardson M , Kreis M , Shewry P R , Halford N G
Published: May 2006

A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisiae) SNF1-encoded protein-serine/threonine kinase, has been isolated from a library prepared from rye endosperm mRNA. Northern blot analysis demonstrated the presence of cRKIN1-related transcripts in developing endosperms but not in shoots, and Southern blot analysis showed the presence of a small gene family. SNF1 plays a central role in carbon catabolite repression in yeast and expression of the RKIN1 sequence in yeast snf1 mutants restored SNF1 function. This suggests that the RKIN1 protein has a role in the control of carbon metabolism in endosperms of rye.
Extensive mitochondrial diversity within a single Amerindian tribe.
Authors: Ward R H , Frazier B L , Dew-Jager K , Pääbo S
Published: May 2006

Sequencing of a 360-nucleotide segment of the mitochondrial control region for 63 individuals from an Amerindian tribe, the Nuu-Chah-Nulth of the Pacific Northwest, revealed the existence of 28 lineages defined by 26 variable positions. This represents a substantial level of mitochondrial diversity for a small local population. Furthermore, the sequence diversity among these Nuu-Chah-Nulth lineages is greater than 60% of the mitochondrial sequence diversity observed in major ethnic groups such as Japanese or sub-Saharan Africans. It was also observed that the majority of the mitochondrial lineages of the Nuu-Chah-Nulth fell into phylogenetic clusters. The magnitude of the sequence difference between the lineage clusters suggests that their origin predates the entry of humans into the Americas. Since a single Amerindian tribe can contain such extensive molecular diversity, it is unnecessary to presume that substantial genetic bottlenecks occurred during the formation of contemporary ethnic groups. In particular, these data do not support the concept of a dramatic founder effect during the peopling of the Americas.
Etoposide (VP-16-213)-induced gene alterations: potential contribution to cell death.
Authors: Berger N A , Chatterjee S , Schmotzer J A , Helms S R
Published: May 2006

We have shown previously a good correlation between etoposide-induced sister chromatid exchanges (SCE) and cytotoxicity. A semisynthetic derivative of podophyllotoxin, etoposide is also called Vepesid (Bristol; code designation VP-16-213, abbreviated VP-16). Since SCE represent DNA recombinational events, we hypothesized that VP-16-induced SCE might result in nonhomologous recombination in which segments of DNA were either deleted or added, leading to an alteration of gene sequences responsible for essential cell proteins. Alterations of such essential genes and consequent interference with formation of their products could consequently lead to cell death. To evaluate whether VP-16 treatment caused sufficient levels of DNA sequence alterations to interfere with gene product formation, we isolated hypoxanthine (guanine) phosphoribosyltransferase (HPRT)-deficient mutants from Chinese hamster V79 cells grown in the presence or absence of VP-16. DNA from 3 spontaneous mutants and 10 VP-16-induced mutants was analyzed by Southern blot hybridization to a full-length hamster HPRT cDNA probe. Most of the VP-16-induced mutants showed partial deletions and/or rearrangements of the HPRT gene. In contrast, spontaneous mutants showed negligible deletions or rearrangements. These results provide strong support for our hypothesis that deletion of genetic sequences may constitute an important component of the mechanism of VP-16-induced cell death.
Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication.
Authors: Romero D L , Busso M , Tan C K , Reusser F , Palmer J R , Poppe S M , Aristoff P A , Downey K M , So A G , Resnick L
Published: May 2006

Certain bis(heteroaryl)piperazines (BHAPs) are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at concentrations lower by 2-4 orders of magnitude than that which inhibits normal cellular DNA polymerase activity. Combination of a BHAP with nucleoside analog HIV-1 RT inhibitors suggested that together these compounds inhibited RT synergistically. In three human lymphocytic cell systems using several laboratory and clinical HIV-1 isolates, the BHAPs blocked HIV-1 replication with potencies nearly identical to those of 3'-azido-2',3'-dideoxythymidine or 2',3'-dideoxyadenosine; in primary cultures of human peripheral blood mononuclear cells, concentrations of these antiviral agents were lower by at least 3-4 orders of magnitude than cytotoxic levels. The BHAPs do not inhibit replication of HIV-2, the simian or feline immunodeficiency virus, or Rauscher murine leukemia virus in culture. Evaluation of a BHAP in HIV-1-infected SCID-hu mice (severe combined immunodeficient mice implanted with human fetal lymph node) showed that the compound could block HIV-1 replication in vivo. The BHAPs are readily obtained synthetically and have been extensively characterized in preclinical evaluations. These compounds hold promise for the treatment of HIV-1 infection.
An unusual mechanism for resistance to the antibiotic coumermycin A1.
Authors: del Castillo I , Vizán J L , Rodríguez-Sáinz M C , Moreno F
Published: May 2006

Bacterial DNA gyrases are type II topoisomerases made up of two A subunits and two B subunits. Coumarins are carbohydrate-containing antibiotics that inhibit topoisomerases II by competing with ATP for binding to the enzymes. High resistance to coumarins is produced in bacterial species by mutations in gyrB, the gene encoding subunit B. We have found an unusual mechanism of resistance to coumarins in Escherichia coli. This mechanism is exhibited by cells containing the wild-type gyrB, or its 5' half, in high copy number. Since homologous mutant gyrB (coumermycin resistant) truncated genes did not confer drug resistance at all under the same conditions, we propose that this mechanism of resistance is due to drug sequestration by the overproduced wild-type GyrB polypeptides. A corollary of this is that the amino half of GyrB is required and sufficient to fashion the ATP-binding domain of DNA gyrase, a conclusion that was further supported by mapping three independent coumarin-resistant mutations at Arg-136 of GyrB. Just upstream of this residue there is a glycine-rich sequence highly conserved in all topoisomerases II, which seems to be a good candidate for the actual ATP-binding site.
Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals.
Authors: Jackson M B , Konnerth A , Augustine G J
Published: May 2006

Hormone release from nerve terminals in the neurohypophysis is a sensitive function of action potential frequency. We have investigated the cellular mechanisms responsible for this frequency-dependent facilitation by combining patch clamp and fluorimetric Ca2+ measurements in single neurosecretory terminals in thin slices of the rat posterior pituitary. In these terminals both action potential-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) and action potential duration were enhanced by high-frequency stimuli, all with a frequency dependence similar to that of hormone release. Furthermore, brief voltage clamp pulses inactivated a K+ current with a very similar frequency dependence. These results support a model for frequency-dependent facilitation in which the inactivation of a K+ current broadens action potentials, leading to an enhancement of [Ca2+]i signals. Further experiments tested for a causal relationship between action potential broadening and facilitation of [Ca2+]i changes. First, increasing the duration of depolarization, either by broadening action potentials with the K(+)-channel blocker tetraethylammonium or by applying longer depolarizing voltage clamp steps, increased [Ca2+]i changes. Second, eliminating frequency-dependent changes in duration, by voltage clamping the terminal with constant duration pulses, substantially reduced the frequency-dependent enhancement of [Ca2+]i changes. These results indicate that action potential broadening contributes to frequency-dependent facilitation of [Ca2+]i changes. However, the small residual frequency dependence of [Ca2+]i changes seen with constant duration stimulation suggests that a second process, distinct from action potential broadening, also contributes to facilitation. These two frequency-dependent mechanisms may also contribute to activity-dependent plasticity in synaptic terminals.
Human growth hormone stimulates proliferation of human retinal microvascular endothelial cells in vitro.
Authors: Rymaszewski Z , Cohen R M , Chomczynski P
Published: May 2006

Growth hormone (GH) has been implicated in the pathogenesis of proliferative diabetic retinopathy. We sought to determine whether this could be mediated by an effect of GH on proliferation of endothelial cells, and, for this purpose, established long-term cultures of human retinal microvascular endothelial cells (hREC) from normal postmortem human eyes. High-purity (greater than 95%) hREC preparations were selected for experiments, based on immunofluorescence with acetylated low density lipoprotein (LDL) and anti-factor VIII-related antigen. Growth requirements for these cells were complex, including serum for maintenance at slow growth rates and additional mitogens for more rapid proliferation. Exposure of hREC to physiologic doses of human GH (hGH) resulted in 100% greater cell number vs. control (P less than 0.01) but could be elicited only in the presence of serum. When differing serum conditions were compared, hGH stimulated [3H]thymidine incorporation up to 1.6- to 2.2-fold under each condition and increased DNA content significantly in the presence of human, horse, and fetal calf serum. Depending on the culture conditions used, the threshold hGH concentration for significant stimulation of hREC proliferation was 0.4-4 micrograms/liter. In contrast, proliferation of human umbilical vein endothelial cells was not significantly altered by hGH added to concentrations as high as 200 micrograms/liter. In summary, hREC respond to physiologic concentrations of hGH in vitro with enhanced proliferation. This specific effect of GH on retinal microvascular endothelial cells supports the hypothesis of a role for GH in endothelial cell biology.
Another discontinuous epitope on glycoprotein gp120 that is important in human immunodeficiency virus type 1 neutralization is identified by a monoclonal antibody.
Authors: Ho D D , Fung M S , Cao Y Z , Li X L , Sun C , Chang T W , Sun N C
Published: May 2006

To define the domains in the envelope glycoprotein important for antibody neutralization of the human immunodeficiency virus type 1 (HIV-1), monoclonal antibodies (mAbs) were generated by immunizing mice with purified glycoprotein gp120 of the IIIB isolate. One mAb, G3-4, reacted with the gp120 of homologous (IIIB) and heterologous (RF) isolates. In addition, mAb G3-4 efficiently neutralized both IIIB and RF viruses in vitro, as well as four of nine primary HIV-1 isolates. In competition immunoassays, mAb G3-4 and soluble CD4 were found to inhibit one another in binding to gp120. However, no competition was seen between mAb G3-4 and mAbs directed to the third variable region or the fourth conserved region of gp120. In particular, mAb G3-4 did not compete with our human mAb 15e, which identifies a discontinuous epitope on gp120 involved in group-specific neutralization of HIV-1 and in gp120-CD4 binding. Epitope-mapping studies on mAb G3-4 with synthetic or unglycosylated recombinant peptides were negative, suggesting that its epitope may be discontinuous. Indeed, this hypothesis was confirmed by showing the loss of mAb G3-4 serologic reactivity when gp120 was first denatured. We conclude that the site recognized by mAb G3-4 represents another discontinuous epitope on gp120 important for neutralization of HIV-1.
A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene.
Authors: O'Neill D , Bornschlegel K , Flamm M , Castle M , Bank A
Published: May 2006

To date, DNA-binding factors with a developmental pattern of expression have not been described in human erythroid cells to explain the switch from fetal (gamma-) to adult (delta- and beta-) globin gene expression. Here we describe a factor present in nuclear extracts from adult mouse and human hematopoietic cells that binds to an oligopyrimidine repeat approximately 960 base pairs upstream from the human delta-globin gene. The binding site for the factor is within an unusual 250-base-pair domain that is greater than 95% pyrimidines on one strand. This domain is preferentially sensitive to S1 nuclease in supercoiled plasmids, indicating that it can adopt an alternative non-B-DNA conformation. A number of S1-sensitive sites within the domain, including the factor-binding site, have sequence characteristics associated with the formation of a triple helix (H-DNA). The position of the binding site between the fetal and adult beta-globin-like genes, its potential for adopting an unusual secondary structure, and the restricted activity of the binding factor to adult hematopoietic tissues suggest possible roles in hematopoietic cell development and hemoglobin switching.
Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.
Authors: Janknecht R , de Martynoff G , Lou J , Hipskind R A , Nordheim A , Stunnenberg H G
Published: May 2006

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.
Phylogenetic and functional affinities of Babakotia (primates), a fossil lemur from northern Madagascar.
Authors: Jungers W L , Godfrey L R , Simons E L , Chatrath P S , Rakotosamimanana B
Published: May 2006

Recent paleontological expeditions to the Ankarana range of northern Madagascar have recovered the partial remains of four individuals of a newly recognized extinct lemur, Babakotia radofilai. Craniodental and postcranial material serve to identify Babakotia as a member of the palaeopropithecids (also including the extinct genera Palaeopropithecus, Archaeoindris, and Mesopropithecus). Living indrids form the sister group to this fossil clade. The postcranial anatomy indicates that Babakotia was a medium-sized (approximately 15 kg) indroid whose inferred positional behaviors were primarily slow climbing and hanging. Although it is probable that a leaping component typified the ancestral positional repertoire of all Malagasy lemurs, the mosaic nature of the locomotor skeleton of Babakotia further suggests that vertical climbing and hang-feeding rather than ricochetal leaping were primitive for indrids and palaeopropithecids and that the dramatic saltatory adaptations of the living indrids postdate the divergence of these two lineages.
Attractive and repulsive interactions between and within adsorbed ribonuclease A layers.
Authors: Belfort G , Lee C S
Published: May 2006

Adsorbed layers of pancreatic RNase A on molecularly smooth mica in aqueous solution attract inorganic mica surfaces whereas they repel similarly adsorbed RNase A layers. As the clean mica surface is covered with RNase A, the attractive interaction slowly diminishes with time and eventually converts to a purely repulsive interaction. Solvent is squeezed out of the solution in the gap during compression of the two surfaces so that the adsorbed protein concentration, as measured directly by the refractive index, increases significantly. The kinetics of this process is analyzed using surface force-distance measurements. All these results are predicted for constrained equilibrium by a discrete lattice model [Scheutjens, J. M. H. M. & Fleer, G. J. (1985) Macromolecules 18, 1882-1900]. Reasonable values are obtained for the constants of the model. We also report on the equilibrium behavior and interaction of densely adsorbed RNase A layers in aqueous solutions of varying ionic strength and pH. With increasing ionic strength, intramolecular forces dominate with diminished electrostatic repulsion. Thus, the adsorbed protein layer becomes more compact while unattached protein molecules coil and fold, making them less likely to form strong intermolecular bridges. Only at very low ionic strength (0.1 mM KCl), when electrostatic forces dominate, does the membrane potential model come close to predicting the long-distance repulsive behavior. Thus, at higher ionic strengths, other non-electrostatic interactions (such as hydrophobic interactions) possibly dominate. An increase in the pH of the solution from 5 to 9.2, the pI of RNase A, significantly reduces the electrostatic repulsion between protein molecules in favor of hydrophobic attractive interactions. This results in lower short-range steric repulsion. However, in contrast to the ionic-strength effect, an increased long-range repulsive force with a much longer decay length is observed. This may be due to contaminants such as DNase that have their pI at a pH other than 9.2. Thus, as with the changing-ionic-strength study, thinner and denser adsorbed layers are formed. Finally, for the kinetic studies, two characteristic length scales--the thickness of the adsorbed layer and the "jump-in" distance--vary linearly with the square root of time. This is consistent with our earlier results and once again implies a diffusion-driven process.
Electrostatic control of midpoint potentials in the cytochrome subunit of the Rhodopseudomonas viridis reaction center.
Authors: Gunner M R , Honig B
Published: May 2006

The photosynthetic reaction center of Rhodopseudomonas viridis has four hemes in a linear arrangement with alternating high- and low-potential sites. Their midpoints are -60, 20, 310, and 380 mV [Dracheva, S. M., Drachev, L. A., Konstantinov, A. A., Semenov, A. Y., Skulachev, V. P., Arutjunjan, A. M., Shuvalov, V. A. & Zaberezhnaya, S. M. (1988) Eur. J. Biochem. 171, 253-264]. Electrostatic calculations reproduce the 440-mV midpoint spread and assignments of high- and low-potential hemes. When calculations on model compounds to connect the theoretical midpoints to the standard hydrogen electrode are used, the absolute electrochemical midpoints for the reaction center hemes are also in good agreement with experiment. The free energy of oxidation is found to be dependent on pairwise interactions with charged amino acids, heme propionic acids, previously oxidized hemes, and axial ligands and on the reaction field induced by heme oxidation.
Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products.
Authors: Ward J E , Dale E M , Binns A N
Published: May 2006

The oriT (origin of transfer) sequence and mob (mobilization) genes of plasmid RSF1010 can functionally replace transfer DNA (T-DNA) borders to generate an RSF1010 intermediate transferable to plants through activities of the tumor-inducing (Ti)-plasmid virulence (vir) genes of Agrobacterium tumefaciens. Because the Ti plasmid virB gene products are hypothesized to form a membrane-localized T-DNA transport apparatus, we investigated whether specific virB genes were needed for RSF1010 transfer. Here we report that transformation of Nicotiana tabacum leaf explants by an RSF1010-derivative plasmid (pJW323) requires the essential virulence genes virB9, virB10, and virB11, consistent with the hypothesis that both the T-DNA and RSF1010 transfer intermediates utilize the same transport machinery. Further, while pJW323 is transferred into plant cells by Agrobacterium strains harboring both pJW323 and pTiA6, the initiation of crown gall tumors (i.e., T-DNA transfer) is greatly suppressed. Coordinate overexpression of the virB9, virB10, and virB11 gene products relieves pJW323-mediated oncogenic suppression and restores tumorigenicity, but does not increase the transfer frequency of pJW323 into plant cells. We propose that the virB9, virB10, and virB11 gene products function coordinately and stoichiometrically to enhance DNA transfer in a fashion specific for the T-DNA intermediate.
Priming of anti-human immunodeficiency virus (HIV) CD8+ cytotoxic T cells in vivo by carrier-free HIV synthetic peptides.
Authors: Hart M K , Weinhold K J , Scearce R M , Washburn E M , Clark C A , Palker T J , Haynes B F
Published: May 2006

The generation of antiviral cytotoxic T lymphocytes (CTLs) is a critical component of the immune response to viral infections. A safe and nontoxic vaccine for AIDS would optimally use a carrier-free synthetic peptide immunogen containing only components of HIV necessary for induction of protective immune responses. We report that hybrid synthetic peptides containing either a HIV envelope gp120 T-cell determinant (T1) or the envelope gp41 fusion domain (F) N-terminal to HIV CTL determinants are capable of priming murine CD8+, major histocompatibility complex class I-restricted anti-HIV CTLs in vivo. These data demonstrate that carrier-free, nonderivatized synthetic peptides can be used in vivo to induce anti-HIV CTL responses.
Colocalization in pericentral hepatocytes in adult mice and similarity in developmental expression pattern of ornithine aminotransferase and glutamine synthetase mRNA.
Authors: Kuo F C , Hwu W L , Valle D , Darnell J E
Published: May 2006

In situ hybridization showed that the mRNA for ornithine aminotransferase (OAT; ornithine-oxo-acid aminotransferase; L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) colocalized with glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] in pericentral hepatocytes of the adult mouse liver. In addition to an identical distribution in adult hepatocytes, OAT and GS have very similar expression patterns in fetal and neonatal liver. As was earlier described for GS, there is a low level of OAT mRNA in fetal cells and increasing pericentral levels in neonates that reach adult patterns within 2 weeks. These results suggest that the transcriptional regulation of the two genes is similar in the liver. However, there was a lack of colocalization of the mRNAs for the two enzymes in cells of the kidney, intestine, and brain, suggesting different regulatory decisions for the OAT and GS genes in the cells of these different tissues. The metabolic consequences of these localized expression patterns favor ammonia clearance from the blood by the liver and urea synthesis by the kidney.
Escape from transforming growth factor beta control and oncogene cooperation in skin tumor development.
Authors: Missero C , Ramon y Cajal S , Dotto G P
Published: May 2006

Control of tumor development by surrounding normal cells has been suggested by a number of in vitro studies. In vivo, tumorigenicity of ras-transformed primary keratinocytes can be suppressed by addition of normal dermal fibroblasts. Here, we report that dermal fibroblasts produce a diffusible inhibitory factor belonging to the transforming growth factor beta (TGF-beta) family and possibly corresponding to TGF-beta 3. This factor can suppress growth of ras-transformed primary keratinocytes in culture and after injection into mice. As with primary cells, tumorigenicity of a ras-transformed, TGF-beta-sensitive keratinocyte line is substantially inhibited by adding dermal fibroblasts, leading to the formation of much smaller and differentiated tumors. Introduction of an intact E1a oncogene into these cells induces concomitant resistance to TGF-beta, to the effect of dermal-fibroblast inhibitory factor, and to dermal-fibroblast tumor suppression. Similar results are obtained with a transformation-deficient truncated E1a mutant, which binds to a reduced subset of cellular proteins (including the retinoblastoma gene product). Thus, genetic events such as those elicited by E1a transformation enable keratinocytes to escape from the inhibitory influences of a normal cellular environment and lead, together with ras transformation, to skin tumor development.
Brain-derived neurotrophic factor: subcellular compartmentalization and interneuronal transfer as visualized with anti-peptide antibodies.
Authors: Wetmore C , Cao Y H , Pettersson R F , Olson L
Published: May 2006

The recent cloning of a second member of the nerve growth factor family, brain-derived neurotrophic factor (BDNF), has prompted investigation into the cells that express this factor's mRNA and protein. In the present study, antibodies raised against unique peptide sequences within the porcine BDNF protein detect BDNF-like immunoreactivity in neurons in rat hippocampal and cortical areas consistent with the distribution of BDNF mRNA as detected with in situ hybridization. Within these neurons, BDNF-like immunoreactivity was observed in the cytoplasm, dendrites, and nuclei. In addition, BDNF immunoreactivity was observed in the cytoplasm of cholinergic neurons that do not express detectable levels of BDNF mRNA. Thus, anti-peptide antibodies can be used to detect this neurotrophic factor protein in cytoplasmic sites of synthesis and in areas of probable action. We propose that one form of the BDNF protein enters the nucleus and may directly influence transcription, while another fraction of the protein is transported out of the synthesizing cell and can be detected, after retrograde axonal transport, in cytoplasmic granules in the perikarya of cholinergic neurons. These basal forebrain cholinergic neurons project to regions enriched in BDNF-synthesizing cells and are known to be responsive to BDNF in vitro. Our data provide information regarding the cellular distribution of BDNF protein in vivo and suggest a dendro-axonic interneuronal transfer of BDNF as well as an additional, intracellular signaling pathway not previously thought to occur in postmitotic neurons in brain.
A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies.
Authors: Zhang B , Roth R A
Published: May 2006

Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding were found to recognize an epitope contained within residues 450-601 of the alpha subunit of the receptor. In contrast, 2 monoclonal antibodies that do not inhibit insulin binding were found to recognize the cysteine-rich region of the alpha subunit. Chimeric insulin receptors that had residues 450-601 replaced with the homologous residues of the insulin-like growth factor I receptor exhibited a decreased ability to bind insulin. In contrast, insulin-like growth factor I receptors that have had the comparable region replaced with that of the insulin receptor showed no decrease in their ability to bind ligand. These results indicate that residues 450-601 of the insulin receptor are important for insulin binding and include the major site for recognition by inhibitory monoclonal antibodies and patients' autoimmune anti-receptor antibodies.
Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.
Authors: Altenhofen W , Ludwig J , Eismann E , Kraus W , Bönigk W , Kaupp U B
Published: May 2006

Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.
Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA.
Authors: Hattman S , Newman L , Murthy H M , Nagaraja V
Published: May 2006

Bacteriophage Mu controls an unusual DNA-modification function encoded by the mom gene, which is located in an operon that consists of two overlapping genes. The com gene, located proximal to the 5' end of the common mRNA transcript, encodes a polypeptide of 62 amino acids that is required for translation of mom. Analysis of the derived amino acid sequence reveals that Com contains zinc-binding finger motifs, suggesting that Com may be a zinc-activated regulatory protein. Atomic absorption analysis showed that there is about one zinc bound per molecule of Com. We have subcloned the com gene into an expression vector and thus have overproduced and purified the Com protein. By gel retardation analysis with various 32P-labeled RNAs (made by in vitro transcription with T7 RNA polymerase), we show that Com binds specifically to com-mom mRNA. A single C----U substitution mutation, located 26 nucleotides upstream from the mom translation start codon, abolishes Com binding. The nature of the Com target sequence was deduced from in vitro footprinting analyses. The results are consistent with the existence of a complex stem-loop structure within the overlap of the com-mom open-reading-frames. Com binding to its target site results in the destabilization of a proposed translation-inhibitor stem-loop (TIS) to expose the Shine-Dalgarno sequence and mom translation initiation codon. This suggests that Com interaction with a specific site on its cognate mRNA alters the mRNA secondary structure to activate translation of mom.
Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines.
Authors: Wilkie T M , Scherle P A , Strathmann M P , Slepak V Z , Simon M I
Published: May 2006

Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.
Low-density particles (W-particles) containing catalase in Zellweger syndrome and normal fibroblasts.
Authors: Aikawa J , Chen W W , Kelley R I , Tada K , Moser H W , Chen G L
Published: May 2006

By both histological and biochemical criteria, peroxisomes in patients with Zellweger syndrome appear to be absent or severely deficient. By using 15-30% (wt/vol) Nycodenz/sucrose gradients to study the subcellular localization of extraperoxisomal catalase activity, a commonly used marker for mature peroxisomes, we detected a single peak of activity in Zellweger syndrome fibroblasts at an equilibrium density of 1.13 g/cm3, lower than the expected 1.17 g/cm3 of mature peroxisomes. Upon recentrifugation in either the original gradient or one with a higher salt concentration, essentially all catalase activity was recovered in fractions of the original densities. The activity of the catalase peak was further analyzed by a digitonin titration and filtration assay in combination with Triton X-100 treatment. The catalase activity passed through 0.1-microns and 0.22-microns but was retained on 0.025-microns membrane filters (mean pore size). After treatment with Triton X-100 nearly all catalase activity passed through the filters. The results from fractionations data, digitonin latency measurement, and the detergent effect on the filtration behavior suggest that catalase is not free in the cytosol of Zellweger syndrome fibroblasts as commonly thought but in particles (W-particles). Similar low-density catalase-containing particles, distinct from peroxisomes, are also found in normal fibroblasts. We found that L-alpha-hydroxyacid oxidase, another peroxisomal matrix enzyme, is also present in W-particles derived from normal and Zellweger syndrome fibroblasts. We speculate that the low-density catalase-containing W-particle may represent an immature or incomplete form of peroxisome distinct from previously described "peroxisomal ghosts" in Zellweger syndrome fibroblasts.
Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates.
Authors: Inchauspe G , Zebedee S , Lee D H , Sugitani M , Nasoff M , Prince A M
Published: May 2006

Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative E1), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.
Cloning and characterization of a 20-kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro.
Authors: Van Nocker S , Vierstra R D
Published: May 2006

Recent evidence indicates that the commitment to degrade cellular proteins by the ubiquitin proteolytic pathway is dependent on the covalent attachment of multiubiquitin chains to the target protein [Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K. & Varshavsky, A. (1989) Science 243, 1576-1583]. We have isolated a 20-kDa ubiquitin carrier protein [E2(20 kDa)] from wheat by using ubiquitin covalent affinity chromatography and anion-exchange HPLC that catalyzes multiubiquitin chain formation in vitro. This reaction is blocked by the addition of a mutant ubiquitin in which arginine has been substituted for lysine at residue 48, demonstrating that the coupling of ubiquitin to ubiquitin is likely to be through an isopeptide linkage between the C-terminal glycine and Lys48 of ubiquitin. By immunoscreening a wheat cDNA expression library with anti-E2(20 kDa) antibodies, a cDNA encoding the complete protein was isolated. The clone (designated UBC7) was confirmed as encoding E2(20 kDa) by comparison of the derived amino acid sequence with peptide sequences of E2(20 kDa) tryptic fragments. The encoded protein contains a single cysteine at position 91, which is presumably the active site, and has regions of amino acid sequence similarity to other known E2s from plants and yeast. Expression of this cDNA in Escherichia coli produced an active E2 capable of catalyzing multiubiquitin chain formation in vitro. By virtue of its activity, E2(20 kDa) may have a pivotal role in protein degradation by the ubiquitin-dependent proteolytic pathway.
A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.
Authors: Runge M S , Quertermous T , Zavodny P J , Love T W , Bode C , Freitag M , Shaw S Y , Huang P L , Chou C C , Mullins D
Published: May 2006

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either beta-globin or mouse immunoglobulin. This modification resulted in a greater than 100-fold improvement in the level of protein expression. The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis. Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.
Delineation of a previously unrecognized cis-acting element required for HLA class II gene expression.
Authors: Sugawara M , Ponath P D , Shin J , Yang Z , Strominger J L
Published: May 2006

The cis-acting DNA sequences that control expression of the HLA-DPA and HLA-DQB promoters have been investigated in detail. A set of recombinant plasmids containing 5' promoter deletions as well as site-directed mutants of the DPA and DQB genes were fused to a reporter gene and transfected into human B and interferon gamma (IFN-gamma)-inducible cells. A previously unrecognized cis element, which is essential both for transcription induced by IFN-gamma in several cell types and for constitutive class II expression in B cells, in addition to the well known X and Y boxes, has been defined. This sequence, which spans nucleotides -107 to -98 and -146 to -137 of the DPA and DQB promoters, respectively, has been called the J element. Some evidence for function of a fourth element, the S element, is also presented.
In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.
Authors: Deforce L , Tomizawa K , Ito N , Farrens D , Song P S , Furuya M
Published: May 2006

Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions. A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated.