bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular dopamine concentration in the retina of the clawed frog, Xenopus laevis.
Authors: Witkovsky P , Nicholson C , Rice M E , Bohmaker K , Meller E
Published: May 2006

Dopamine reaches targets in the outer retina of the clawed frog (Xenopus laevis) by diffusion from a network of dopaminergic cells and processes located predominantly at the junction of inner nuclear and inner plexiform layers. We obtained values for the steady-state release, uptake, and extracellular concentration of dopamine in the retina by a combination of HPLC (with electrochemical detection), scintillation spectroscopy, and fast-scan cyclic voltammetry. Vitreal concentrations of dopamine varied from 564 +/- 109 nM in light-adapted eyes near the time of subjective dawn to 156 +/- 12 nM in dark-adapted eyes. The data are consistent with a simple model for steady-state dopamine diffusion from an appropriately sited thin-sheet source. This model was used to generate a profile of extracellular dopamine concentration as a function of retinal depth. The model predicted an increase in the dopamine concentration from the vitreous to the layer of dopaminergic cells, remaining constant from that layer to the distal tips of the photoreceptors. This prediction was borne out by comparing fast-scan voltammetric measures of dopamine at the distal tips of the receptors with the vitreal concentrations determined by HPLC using electrochemical detection.
Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation.
Authors: May W A , Gishizky M L , Lessnick S L , Lunsford L B , Lewis B C , Delattre O , Zucman J , Thomas G , Denny C T
Published: May 2006

The 11;22 chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminal-encoding portion of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI1 gene. We have isolated a fourth EWS-FLI1 fusion cDNA that is structurally distinct from the three forms previously described. To determine the transforming activity of this gene, alternative forms of the EWS-FLI1 fusion were transduced into NIH 3T3 cells. Cells expressing either type 1 or type 4 fusion constructs formed foci in culture and colonies in soft agar, indicating that EWS-FLI1 is a transforming gene. EWS-FLI1 deletion mutants were created to map functionally the critical regions within the chimera. Deletion of either the EWS domain or the FLI1 corresponding to the DNA-binding domain totally abrogated the ability for EWS-FLI1 to transform 3T3 cells. These data indicate that the oncogenic effect of the 11;22 translocation is caused by the formation of a chimeric transcription factor. Formation of chimeric transcription factors has now been demonstrated to promote tumors of both neuroectodermal and hematopoietic origin, suggesting that this may be a common mechanism in human carcinogenesis.
Nerve growth factor induces neuron-like differentiation of an insulin-secreting pancreatic beta cell line.
Authors: Polak M , Scharfmann R , Seilheimer B , Eisenbarth G , Dressler D , Verma I M , Potter H
Published: May 2006

Nerve growth factor (NGF) is the best understood of a class of trophic proteins that are important for the survival of neurons and the elaboration of their characteristic processes. Here we demonstrate that RINm5F, a rat insulinoma cell line representing an early stage in pancreatic beta cell differentiation, expresses both the Trk and p75 NGF receptors and responds to NGF by extending neurite-like (neurofilament-containing) processes. NGF treatment of RINm5F cells also induces the expression of genes normally responsive to NGF in neurons, including the NGF-1A gene. Inasmuch as pancreatic beta cells arise from the embryonic endoderm, these results suggest that NGF may play a wider role during development than previously thought-a role not restricted to cells of neuroectodermal origin--and that endocrine and neuronal cells share a developmental pathway. The specific effect of NGF on an early pancreatic beta cell line also suggests that this neurotrophic factor might form the basis of a therapeutic treatment for some types of diabetes by inducing the proliferative differentiation of islet cells.
Nonexponential relaxation after ligand dissociation from myoglobin: a molecular dynamics simulation.
Authors: Kuczera K , Lambry J C , Martin J L , Karplus M
Published: May 2006

Molecular dynamics simulations of myoglobin after ligand photodissociation show that the out-of-plane motion of the heme iron has a rapid subpicosecond phase followed by a slower nonexponential process involving more global protein relaxation. Individual trajectories show rather different behavior, suggesting there is an inhomogeneous component to the relaxation. The calculated time dependence of the iron motion over 100 ps is in excellent agreement with the frequency shift of band III of the heme group [see Lim, M., Jackson, T. A. & Anfinrud, P. A. (1993) Proc. Natl. Acad. Sci. USA 90, 5801-5804]. If that the barrier to rebinding depends on the out-of-plane iron position, the time dependence obtained from the simulation can explain the nonexponential room-temperature geminate recombination of NO.
Identification and functional characterization of platelet-activating factor receptors in human leukocyte populations using polyclonal anti-peptide antibody.
Authors: Müller E , Dagenais P , Alami N , Rola-Pleszczynski M
Published: May 2006

Recently, the successful cloning of a receptor for platelet-activating factor (PAF), a lipid mediator of inflammation, was reported. Here we investigated the distribution and potential diversity of human PAF receptors (hPAF-Rs) among individual leukocyte populations by (i) hPAF-R mRNA transcription studies and (ii) analysis of cell surface expression of hPAF-R protein using a polyclonal anti-peptide antibody (anti-hPAF-R164-173). Northern blot analysis, flow cytometry, and immunoblotting with anti-hPAF-R antibody indicated that monocytic, neutrophilic, and B-lymphocytic cell lines all shared a similar hPAF-R species, whereas resting T-cell and natural killer cell lines failed to express detectable levels of either hPAF-R protein or mRNA. Peripheral blood leukocyte populations showed a distribution of hPAF-R cell surface expression similar to that of the corresponding cell lines. Furthermore, binding of anti-hPAF-R164-173 antiserum, purified IgG, or Fab and F(ab')2 fragments to the receptor of all investigated PAF-R-positive cell lines induced an increase in intracellular free calcium concentration. The characterization of the expression of a lipid ligand receptor using antibodies against an intrinsic portion of the receptor protein has, to our knowledge, never been reported previously.
Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization.
Authors: Robinson J , Schmitt E A , Hárosi F I , Reece R J , Dowling J E
Published: May 2006

In many vertebrates, UV-sensitive photoreceptors have been identified by microspectrophotometry and UV-visual sensitivity has been identified by behavioral studies, but as yet no vertebrate UV-sensitive pigment gene has been isolated. We have sequenced a cDNA clone that hybridizes to short single cone cells in the zebrafish (Brachydanio rerio). These cells, which make up 25% of the cone population in zebrafish retinae, are UV-sensitive (lambda max approximately 360 nm). The visual pigment encoded by this gene is unusual in that its amino acid sequence is more homologous to the rod pigment rhodopsin (up to 89%) than to other cone pigments (35-83%). Like all other vertebrate visual pigments, it contains a lysine residue at position 296, the presumptive retinal binding site, and a glutamate residue at position 113. However, it is unique in possessing a lysine residue at position 126, which may account for the UV-sensitivity of the pigment.
A putative Wilms tumor-secreted growth factor activity required for primary culture of human nephroblasts.
Authors: Burrow C R , Wilson P D
Published: May 2006

Establishment of cell culture systems for the study of organogenesis during human embryonic development could provide the basis for the study of molecular mechanisms that regulate cellular proliferation and organ morphogenesis. We have developed a cell culture system for undifferentiated mesenchymal cells isolated from the human fetal kidney, which retain the potential for conversion to differentiated epithelia in vitro. Microdissected marginal zone nephroblasts were treated with trypsin and plated on gelatin prior to unlimited serial passage in suspension. An absolute requirement for the indefinite proliferation of these undifferentiated progenitors was nephroblast growth factor (NB-GF), a growth factor activity secreted by a Wilms tumor cell line. The mitogenic effects of NB-GF were not reproduced by previously described growth factors known to be mitogenic for renal cells or by leukemia inhibitory factor. In addition, cultured nephroblasts were shown to retain their ability to differentiate into epithelia when exposed to 10% serum-containing medium in the absence of NB-GF. Immunocytochemical cytoskeletal protein marker analysis showed mutually exclusive staining of vimentin in nephroblasts and cytokeratin in epithelia. These findings suggest that NB-GF may play an important role in the regulation of nephroblast proliferation during renal development and in Wilms tumor biology.
Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase.
Authors: Berry A M , Harriott O T , Moreau R A , Osman S F , Benson D R , Jones A D
Published: May 2006

Biological nitrogen fixation in aerobic organisms requires a mechanism for excluding oxygen from the site of nitrogenase activity. Oxygen exclusion in Frankia spp., members of an actinomycetal genus that forms nitrogen-fixing root-nodule symbioses in a wide range of woody Angiosperms, is accomplished within specialized structures termed vesicles, where nitrogen fixation is localized. The lipidic vesicle envelope is apparently a functional analogue of the cyanobacterial heterocyst envelope, forming an external gas-diffusion barrier around the nitrogen-fixing cells. We report here that purified vesicle envelopes consist primarily of two hopanoid lipids, rather than of glycolipids, as is the case in cyanobacteria. One envelope hopanoid, bacteriohopanetetrol phenylacetate monoester, is vesicle-specific. The Frankia vesicle envelope thus represents a layer specific to the locus of nitrogen fixation that is biosynthetically uniquely derived.
Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination.
Authors: Silver D P , Spanopoulou E , Mulligan R C , Baltimore D
Published: May 2006

As a probe of whether RAG-1 and RAG-2 gene products activate other genes or form part of the recombinase itself, certain mutants of the RAG genes were assayed for their ability to activate variable-diversity-joining region [V(D)J] recombination in a plasmid substrate in fibroblasts. The results indicate that the N-terminal one-third of RAG-1, including a zinc-finger-like domain, and an acidic domain of RAG-2 are dispensable for activating V(D)J recombination in a fibroblast, although they contribute quantitatively. In contrast, deletion of the C-terminal segment of RAG-1, which has homology to a topoisomerase-like protein from yeast, abolished recombination activation. These results do not support the hypothesis that the RAG gene products are transcription factors and suggest the possibility that they are parts of the recombination machinery.
Several short interspersed repetitive elements (SINEs) in distant species may have originated from a common ancestral retrovirus: characterization of a squid SINE and a possible mechanism for generation of tRNA-derived retroposons.
Authors: Ohshima K , Koishi R , Matsuo M , Okada N
Published: May 2006

Using labeled transcripts generated in vitro from squid total genomic DNA as a probe, we isolated and characterized a SINE that is present in the squid genome. The squid SINE appears to be derived from a tRNA(Lys). When the consensus sequences of five different SINEs with a tRNA(Lys)-like structure from distantly related species, including squid, were aligned, we found in the tRNA-unrelated region two sequence motifs that were almost identical among these five SINEs. This observation suggests a common evolutionary origin for these SINEs and/or some function(s) for these motifs. Similar sequences were unexpectedly found to be present in sequences complementary to the U5 regions of several mammalian retroviruses whose primer is a tRNA(Lys). On the basis of these findings, we present a model for the generation of SINEs. We propose that they are derived from a "strong-stop DNA" with a primer tRNA(Lys) that is an intermediate in the reverse transcription of certain retroviruses. Our model suggests that a certain group of SINEs may have been generated by horizontal transmission, although it is not clear whether information was transmitted via a similar retrovirus or via an RNA or DNA of a SINE.
Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils.
Authors: Hamada F , Aoki M , Akiyama T , Toyoshima K
Published: May 2006

The interaction of Fc receptors with antibody-antigen complexes activates multiple biological functions in hematopoietic cells. Recently, protein-tyrosine phosphorylation has been suggested to be involved in Fc receptor-mediated cell signaling. Here we show that the Src-like protein-tyrosine kinase Fgr, which is specifically expressed in mature myelomonocytic cells, coimmunoprecipitates with IgG Fc receptor II (Fc gamma RII), but not with Fc gamma RIII from detergent lysates of human peripheral neutrophils. Crosslinking of Fc gamma RII induced a rapid increase in the tyrosine kinase activity and comodulation of Fgr. These results suggest that Fgr is physically and functionally associated with Fc gamma RII and involved in Fc gamma RII-mediated signal transduction pathways.
Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.
Authors: Jacobo-Molina A , Ding J , Nanni R G , Clark A D , Lu X , Tantillo C , Williams R L , Kamer G , Ferris A L , Clark P , Hizi A , Hughes S H , Arnold E
Published: May 2006

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.
Kinetics and thermodynamics of folding in model proteins.
Authors: Camacho C J , Thirumalai D
Published: May 2006

Monte Carlo simulations on a class of lattice models are used to probe the thermodynamics and kinetics of protein folding. We find two transition temperatures: one at T theta, when chains collapse from a coil to a compact phase, and the other at Tf (< T theta), when chains adopt a conformation corresponding to their native state. The kinetics are probed by several correlation functions and are interpreted in terms of the underlying energy landscape. The transition from the coil to the native state occurs in three distinct stages. The initial stage corresponds to a random collapse of the protein chain. At intermediate times tau c, during which much of the native structure is acquired, there are multiple pathways. For longer times tau r (>> tau c) the decay is exponential, suggestive of a late transition state. The folding time scale (approximately tau r) varies greatly depending on the model. Implications of our results for in vitro folding of proteins are discussed.
Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.
Authors: Dubois B W , Cherian S F , Evers A S
Published: May 2006

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein.
Short DNA sequences from the cytoplasm of mouse tumor cells induce immortalization of human lymphocytes in vitro.
Authors: Abken H , Hegger R , Bützler C , Willecke K
Published: May 2006

Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA sequences that induce unlimited proliferation ("immortalization") of human lymphocytes after transfection in vitro. By equilibrium centrifugation of cytoplasmic lysates in a neutral CsCl gradient, the immortalizing activity was recovered together with extramitochondrial fractions at high salt densities (1.85-1.87 g/cm3). Unexpectedly, these fractions contain linear DNA molecules of 50-500 bp in length. In contrast, cytoplasts of primary, senescent cells (mouse embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding fractions. Cytoplasmic DNA isolated from high-density fractions of mouse tumor cells was cloned in subset libraries, and of 45 DNA sequences we identified 2 clones--one from L929 cytoplasts (203 bp) and another one from the cytoplasm of Ehrlich ascites cells (372 bp)--that induce unlimited proliferation of human lymphocytes in vitro. Immortalized lymphoid cells harbor 1-5 copies of transfected DNA integrated into chromosomal DNA, whereas about 100 copies were found as episomal DNA in the cytoplasmic fraction. No immortalization could be induced by transfection of nuclear DNA randomly fragmented to 200-500 bp. Although the cloned DNA sedimented at 1.70 g/cm3, after transient transfection into lymphocytes, these DNA sequences form salt-stable complexes that sediment in fractions at the same high density (1.82-1.88 g/cm3) from which they were originally cloned. The high-density banding of these cytoplasmic DNA sequences may be due to association with RNA and/or with (metallo-) proteins in vivo. Since both cloned DNA sequences with immortalizing activity have stop codons for protein translation in all possible reading frames, immortalization may be induced by insertional inactivation or functional suppression of genes that are needed to be expressed during cellular senescence or programmed cell death.
Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae.
Authors: White M A , Dominska M , Petes T D
Published: May 2006

The full activity of a recombination initiation site located 5' of HIS4 requires the binding of the transcription factors RAP1, BAS1, and BAS2. Two RAP1 binding sites can substitute for the wild-type initiation site. A 51-bp region of telomeric DNA inserted upstream of either HIS4 or ARG4 very strongly stimulates recombination. We suggest that the ability of transcription factors to induce recombination is a consequence of an altered chromatin structure that favors the entry of proteins that initiate recombination, rather than an effect of these factors on transcription.
Human immunodeficiency virus proteins induce the inhibitory cAMP/protein kinase A pathway in normal lymphocytes.
Authors: Hofmann B , Nishanian P , Nguyen T , Insixiengmay P , Fahey J L
Published: May 2006

Proliferation of normal T lymphocytes is impaired by human immunodeficiency virus (HIV) proteins. In this paper, we demonstrate important parts of this mechanism. Initially, HIV-induced impairment of proliferation was shown to be an active process involving induction of protein tyrosine kinases in both CD4 and CD8 T cells. Furthermore, the impairment of cell proliferation was demonstrated to be linked to induction of the inhibitory protein kinase A (PKA) pathway by HIV proteins. This induction of PKA was accompanied by an increase in intracellular cAMP, which is necessary for the activation of PKA. Finally, increases in cAMP/PKA activity were shown to induce biochemical changes that impaired proliferation when cells were stimulated with phytohemagglutinin. This was demonstrated by showing that (i) agents, other than HIV proteins, that increase cAMP/PKA activity (cholera toxoid and 8-bromo-cAMP) also decreased T-lymphocyte proliferation; (ii) exposure of lymphocytes to HIV or cholera toxoid led to decreased membrane activity of the proliferation promoter protein kinase C upon stimulation; and (iii) agents that reduced cAMP generation neutralized the effect of HIV proteins and restored lymphocyte proliferation. These studies show that the HIV-induced augmentation of cAMP/PKA activity may be a key part of the mechanism responsible for all or part of the HIV-induced anergy of T lymphocytes.
Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.
Authors: Jiang C K , Magnaldo T , Ohtsuki M , Freedberg I M , Bernerd F , Blumenberg M
Published: May 2006

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.
Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii.
Authors: Oelschlaeger T A , Guerry P , Kopecko D J
Published: May 2006

Bacterial invasion of six different human epithelial cell lines showed that some strains of the intestinal pathogen Campylobacter jejuni invaded intestinal cell lines at a level 10(2)-10(4) times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacter jejuni strain 81-176 uptake into 407 intestinal cells and Citrobacter entry into T24 bladder cells was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microfilament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacter jejuni or Citrobacter freundii, but monensin markedly reduced Citrobacter uptake. Epithelial cell invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cell protein synthesis. In contrast to the T24 cell line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule- and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfilament-dependent) that are cell line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.
Determination of the phylogenetic relationships among Pacific salmonids by using short interspersed elements (SINEs) as temporal landmarks of evolution.
Authors: Murata S , Takasaki N , Saitoh M , Okada N
Published: May 2006

Several subfamilies of the salmonid Hpa I short interspersed element (SINE) family were isolated from salmonid genomes and were sequenced. For each genomic locus that represented the subfamily, amplification by PCR of the orthologous loci in the 12 fish allowed us to determine the order of branching of the Pacific salmonid species. The deduced phylogeny suggests three evolutionary lines, namely, a line of chum salmon, pink salmon, and kokanee; a line of coho salmon and chinook salmon; and a line of steelhead trout. Our data also support a change in the phylogenetic assignment of steelhead trout from Salmo to Oncorhynchus. We present here an extensive phylogenetic tree constructed from an analysis of differential insertion of SINEs, and we propose that SINE insertion analysis is one of the best available methods for clarifying the order of divergence of closely related species.
Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases.
Authors: Wilson C , Goberdhan D C , Steller H
Published: May 2006

We have identified a Drosophila gene, Dror, which encodes a putative receptor tyrosine kinase (RTK) and maps to cytological location 31B/C on the second chromosome. In embryos, this gene is expressed specifically in the developing nervous system. The Dror protein appears to be a homolog of two human RTKs, Ror1 and Ror2. Dror and Ror1 proteins share 36% amino acid identity in their extracellular domains and 61% identity in their catalytic tyrosine kinase (TK) domains. Ror1 and Ror2 were originally identified on the basis of the similarity of their TK domains to the TK domains of members of the Trk family of neurotrophin receptors. The Dror protein shows even greater similarity to the Trk proteins within this region than do the human Ror proteins. In light of its similarity to trk and its neural-specific expression pattern, we suggest that Dror may encode a neurotrophic receptor that functions during early stages of neural development in Drosophila.
High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.
Authors: Lufkin T , Lohnes D , Mark M , Dierich A , Gorry P , Gaub M P , LeMeur M , Chambon P
Published: May 2006

Retinoic acid (RA) plays a critical role in normal development, growth, and maintenance of certain tissues. The action of RA is thought to be mediated in part by the three nuclear receptors (RAR alpha, -beta, and -gamma), each of which is expressed as multiple isoforms. To investigate the function of the RAR alpha gene, we have disrupted, in the mouse, the whole gene or the isoform RAR alpha 1. Although RAR alpha 1 is the predominant isoform and is highly conserved among vertebrates, RAR alpha 1-null mice appeared normal. However, targeted disruption of the whole RAR alpha gene resulted in early postnatal lethality and testis degeneration. These results, showing that RAR alpha is indeed involved in the transduction of the RA signal, also suggest an unexpected genetic redundancy.
Nitric oxide activates cyclooxygenase enzymes.
Authors: Salvemini D , Misko T P , Masferrer J L , Seibert K , Currie M G , Needleman P
Published: May 2006

We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 microgram/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine. The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1 beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1 beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.
The synonymous substitution rate of the major histocompatibility complex loci in primates.
Authors: Satta Y , O'hUigin C , Takahata N , Klein J
Published: May 2006

Because the divergence of many allelic lineages at the major histocompatibility complex (MHC) loci predates species divergence, standard methods of calculating synonymous substitution rates are not applicable to this system. We used three alternative methods of rate estimation: one based on the minimum number of substitutions (Dm), another on the nucleotide difference (Dxy), and the third on the net nucleotide difference (Dn). We applied these methods to the protein-encoding sequences of primate MHC class I (A, B, and C) and class II (DRB1) genes. To determine the reliability of the different estimates, we carried out computer simulation. The distribution of the estimates based on Dxy or Dn is generally much broader than that based on Dm. More importantly, the Dm-based method nearly always has the highest probability of recovering true rates, provided that Dm is not smaller than 5. Because of its desirable statistical properties, we used the Dm-based method to estimate the rate of synonymous substitutions. The rate is 1.37 +/- 0.61 for A, 1.84 +/- 0.40 for B, 3.87 +/- 1.05 for C, and 1.18 +/- 0.36 for DRB1 loci, always per site per 10(9) years. Hence despite the extraordinary polymorphism, the mutation rate at the primate MHC loci is no higher than that of other loci.
A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.
Authors: Brinkmann U , Reiter Y , Jung S H , Lee B , Pastan I
Published: May 2006

B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFV)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.
Improved prediction of protein secondary structure by use of sequence profiles and neural networks.
Authors: Rost B , Sander C
Published: May 2006

The explosive accumulation of protein sequences in the wake of large-scale sequencing projects is in stark contrast to the much slower experimental determination of protein structures. Improved methods of structure prediction from the gene sequence alone are therefore needed. Here, we report a substantial increase in both the accuracy and quality of secondary-structure predictions, using a neural-network algorithm. The main improvements come from the use of multiple sequence alignments (better overall accuracy), from "balanced training" (better prediction of beta-strands), and from "structure context training" (better prediction of helix and strand lengths). This method, cross-validated on seven different test sets purged of sequence similarity to learning sets, achieves a three-state prediction accuracy of 69.7%, significantly better than previous methods. In addition, the predicted structures have a more realistic distribution of helix and strand segments. The predictions may be suitable for use in practice as a first estimate of the structural type of newly sequenced proteins.
Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes.
Authors: Scherle P , Behrens T , Staudt L M
Published: May 2006

The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.
Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.
Authors: Felder C C , Briley E M , Axelrod J , Simpson J T , Mackie K , Devane W A
Published: May 2006

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.
Kinetoplast DNA maxicircles: networks within networks.
Authors: Shapiro T A
Published: May 2006

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is an enormous network of interlocked minicircles and maxicircles. We selectively removed minicircles from Trypanosoma equiperdum kDNA networks by restriction enzyme cleavage. Maxicircles remained in aggregates that were resistant to protease or RNase and contained no residual minicircles, but were resolved into circular monomers by topoisomerase II. Maxicircles thus form independent catenanes within kDNA networks. Heterogeneity in the size, composition, and organization of maxicircle catenanes reflects changes that occur during kDNA replication. The rosette-like arrangement of maxicircle catenanes is distinctly different from that of minicircle catenanes. Trypanosome kDNA networks reveal unique topological complexity: they are composed of entirely dissimilar catenanes that are in turn extensively interlocked with one another.
The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumor suppressor protein of septate junctions.
Authors: Willott E , Balda M S , Fanning A S , Jameson B , Van Itallie C , Anderson J M
Published: May 2006

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1, the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa are homologous to the product of the lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, located in septate junctions, and to a 95-kDa protein located in the postsynaptic densities of rat brain, PSD-95. All three proteins contain both a src homology region 3 (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression was previously shown to correlate with variable properties of tight junctions. dlg mutations result in loss of apical-basolateral epithelial cell polarity and in neoplastic growth. These results suggest a protein family specialized for signal transduction on the cytoplasmic surface of intercellular junctions. These results also provide biochemical evidence for similarity between invertebrate septate and vertebrate tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions.
Oxidants, antioxidants, and the degenerative diseases of aging.
Authors: Ames B N , Shigenaga M K , Hagen T M
Published: May 2006

Metabolism, like other aspects of life, involves tradeoffs. Oxidant by-products of normal metabolism cause extensive damage to DNA, protein, and lipid. We argue that this damage (the same as that produced by radiation) is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts. Antioxidant defenses against this damage include ascorbate, tocopherol, and carotenoids. Dietary fruits and vegetables are the principal source of ascorbate and carotenoids and are one source of tocopherol. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake and also markedly increases the risk of heart disease and cataracts. Since only 9% of Americans eat the recommended five servings of fruits and vegetables per day, the opportunity for improving health by improving diet is great.
Selective proteolytic activity of the antitumor agent kedarcidin.
Authors: Zein N , Casazza A M , Doyle T W , Leet J E , Schroeder D R , Solomon W , Nadler S G
Published: May 2006

Kedarcidin is a potent antitumor antibiotic chromoprotein, composed of an enediyne-containing chromophore embedded in a highly acidic single chain polypeptide. The chromophore was shown to cleave duplex DNA site-specifically in a single-stranded manner. Herein, we report that in vitro, the kedarcidin apoprotein, which lacks any detectable chromophore, cleaves proteins selectively. Histones that are the most opposite in net charge to the apoprotein are cleaved most readily. Our findings imply that the potency of kedarcidin results from the combination of a DNA damaging-chromophore and a protease-like apoprotein.
Ectopic neural expression of a floor plate marker in frog embryos injected with the midline transcription factor Pintallavis.
Authors: Ruiz i Altaba A , Cox C , Jessell T M , Klar A
Published: May 2006

The floor plate, a cell group that develops at the midline of the neural plate in response to inductive signals from the notochord, has been implicated in the control of dorsoventral neural pattern. The frog Pintallavis gene, encoding a member of the HNF-3/fork head transcription factor family, is expressed in the notochord and in midline neural plate cells that give rise to the floor plate. To examine whether Pintallavis might be involved in regulating the differentiation of the floor plate, we ectopically expressed Pintallavis by injection of synthetic mRNA into two-cell frog embryos. Injection of Pintallavis mRNA resulted in the ectopic expression of F-spondin, a gene encoding a floor plate-specific adhesion molecule, at the dorsal midline of the neural tube. The expression of Pintallavis in midline cells may therefore contribute to the establishment of the floor plate fate.
Cooperative and anticooperative binding to a ribozyme.
Authors: Bevilacqua P C , Johnson K A , Turner D H
Published: May 2006

The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15 degrees C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate. Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.