bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiparticle adhesive dynamics: Hydrodynamic recruitment of rolling leukocytes
Authors: King Michael R. , Hammer Daniel A.
Published: July 2002

The slow rolling motion of leukocytes along the walls of
blood vessels mediated by specific receptor-ligand adhesion is
important in inflammation and occurs in postcapillary venules over a
wide range of wall shear stresses and vessel diameters. The ability of
hydrodynamic collisions between cells to induce capture of free-stream
leukocytes to a selectin-bearing surface under shear flow was studied
experimentally by using a cell-free assay. It was found that
carbohydrate-coated spherical beads, representing model leukocytes,
tend to attach to the adhesive wall 4–5 cell diameters up- or
downstream of a slowly rolling or stationary adhesive bead. A key
feature of such “hydrodynamic recruitment” is that only glancing,
indirect collisions occurring close to the plane will result in
downstream attachment. A direct numerical simulation of cell capture
and rolling that includes multiparticle hydrodynamic interactions is
shown to reproduce the observed behavior accurately. The theory
predicts that hydrodynamic recruitment will occur in the absence of
buoyancy effects and over a range of shear rates, suggesting that the
mechanism may be important
in vivo
. This theory
is supported by measurements of leukocyte capture
in
vivo
using the hamster cheek pouch model.
DNA-PKcs is critical for telomere capping
Authors: Gilley David , Tanaka Hiromi , Hande M. Prakash , Kurimasa Akihiro , Li Gloria C. , Oshimura Mitsuo , Chen David J.
Published: July 2002

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is
critical for DNA repair via the nonhomologous end joining
pathway. Previously, it was reported that bone marrow cells and
spontaneously transformed fibroblasts from SCID (severe combined
immunodeficiency) mice have defects in telomere maintenance. The
genetically defective SCID mouse arose spontaneously from its parental
strain CB17. One known genomic alteration in SCID mice is a truncation
of the extreme carboxyl terminus of DNA-PKcs, but other as yet
unidentified alterations may also exist. We have used a defined system,
the DNA-PKcs knockout mouse, to investigate specifically the role
DNA-PKcs specifically plays in telomere maintenance. We report that
primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney
cells from 6–8 month-old DNA-PKcs-deficient mice accumulate a large
number of telomere fusions, yet still retain wild-type telomere length.
Thus, the phenotype of this defect separates the two-telomere related
phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs
also exhibit elevated levels of chromosome fragments and breaks, which
correlate with increased telomere fusions. Based on the high levels of
telomere fusions observed in DNA-PKcs deficient cells, we conclude that
DNA-PKcs plays an important capping role at the mammalian telomere.
Mutational analysis and molecular modeling of the nonapeptide hormone binding domains of the [Arg8]vasotocin receptor.
Authors: Hausmann H , Richters A , Kreienkamp H J , Meyerhof W , Mattes H , Lederis K , Zwiers H , Richter D
Published: July 2002

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H)Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.
Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.
Authors: Miranda L , Wolf J , Pichuantes S , Duke R , Franzusoff A
Published: July 2002

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.
Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.
Authors: Efthimiopoulos S , Vassilacopoulou D , Ripellino J A , Tezapsidis N , Robakis N K
Published: July 2002

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.
Brain lipids that induce sleep are novel modulators of 5-hydroxytrypamine receptors.
Authors: Huidobro-Toro J P , Harris R A
Published: July 2002

Amide derivatives of fatty acids were recently isolated from cerebrospinal fluid of sleep-deprived animals and found to induce sleep in rats. To determine which brain receptors might be sensitive to these novel neuromodulators, we tested them on a range of receptors expressed in Xenopus oocytes. cis-9,10-Octadecenamide (ODA) markedly potentiated the action of 5-hydroxytryptamine (5-HT) on 5-HT2A and 5-HT2C receptors, but this action was not shared by related compounds such as oleic acid and trans-9,10-octacenamide. ODA was active at concentrations as low as 1 nM. The saturated analog, octadecanamide, inhibited rather than potentiated 5-HT2C responses. ODA had either no effect or only weak effects on other receptors, including muscarinic cholinergic, metabotropic glutamate, GABA(A), N-methyl-D-asparate, or alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid receptors. Modulation of 5-HT2 receptors by ODA and related lipids may represent a novel mechanism for regulation of receptors that activate G proteins and thereby play a role in alertness, sleep, and mood as well as disturbances of these states.
Dual signal transduction through delta opioid receptors in a transfected human T-cell line.
Authors: Sharp B M , Shahabi N A , Heagy W , McAllen K , Bell M , Huntoon C , McKean D J
Published: July 2002

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.
The [(G/C)3NN]n motif: a common DNA repeat that excludes nucleosomes.
Authors: Wang Y H , Griffith J D
Published: July 2002

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3 NN3']n motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)48 block is 4.9 +/- 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and approximately 78-fold less efficient than a similar length (CTG)n sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of "TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites.
Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.
Authors: Nelles E , Bützler C , Jung D , Temme A , Gabriel H D , Dahl U , Traub O , Stümpel F , Jungermann K , Zielasek J , Toyka K V , Dermietzel R , Willecke K
Published: July 2002

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.
The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.
Authors: Whitchurch C B , Alm R A , Mattick J S
Published: July 2002

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.
Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.
Authors: Neeleman A P , van de Eijnden D H
Published: July 2002

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.
Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.
Authors: Vidal M , Braun P , Chen E , Boeke J D , Harlow E
Published: July 2002

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.
Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.
Authors: Ericsson J , Jackson S M , Lee B C , Edwards P A
Published: July 2002

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.
Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23).
Authors: Daniel D , Wegmann D R
Published: July 2002

The observation that overt type I diabetes is often preceded by the appearance of insulin autoantibodies and the reports that prophylactic administration of insulin to biobreeding diabetes-prone (BB-DP) rats, nonobese diabetic (NOD) mice, and human subjects results in protection from diabetes suggest that an immune response to insulin is involved in the process of beta cell destruction. We have recently reported that islet-infiltrating cells isolated from NOD mice are enriched for insulin-specific T cells, that insulin-specific T cell clones are capable of adoptive transfer of diabetes, and that epitopes present on residues 9-23 of the B chain appear to be dominant in this spontaneous response. In the experiments described in this report, the epitope specificity of 312 independently isolated insulin-specific T cell clones was determined and B-(9-23) was found to be dominant, with 93% of the clones exhibiting specificity toward this peptide and the remainder to an epitope on residues 7-21 of the A chain. On the basis of these observations, the effect of either subcutaneous or intranasal administration of B-(9-23) on the incidence of diabetes in NOD mice was determined. The results presented here indicate that both subcutaneous and intranasal administration of B-(9-23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830-843). This protective effect is associated with reduced T-cell proliferative response to B-(9-23) in B-(9-23)-treated mice.
Identification of the endothelial cell binding site for factor IX.
Authors: Cheung W F , van den Born J , Kühn K , Kjellén L , Hudson B G , Stafford D W
Published: July 2002

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.
Proton long-range migration along protein monolayers and its consequences on membrane coupling
Authors: Gabriel B. , Teissié J.
Published: July 2002

It has been shown with lipid layers and more recently with purple
membranes that protons have slow surface-to-bulk transfer. This results
in long-range proton lateral conduction along membranes. We report here
that such lateral transfer can take place along a pure protein film. It
is strongly controlled by the packing. Subtle reorganizations of the
protein–protein contact can be biological switches between interfacial
and delocalized proton pathways between sources and sinks.
Correction of obesity and diabetes in genetically obese mice by leptin gene therapy
Authors: Muzzin Patrick , Eisensmith Randy C. , Copeland Kenneth C. , Woo Savio L. C.
Published: July 2002

The
ob
/
ob
mouse is genetically
deficient in leptin and exhibits both an obese and a mild
non-insulin-dependent diabetic phenotype. To test the hypothesis that
correction of the obese phenotype by leptin gene therapy will lead to
the spontaneous correction of the diabetic phenotype, the
ob
/
ob
mouse was treated with a
recombinant adenovirus expressing the mouse leptin cDNA. Treatment
resulted in dramatic reductions in both food intake and body weight, as
well as the normalization of serum insulin levels and glucose
tolerance. The subsequent diminishment in serum leptin levels resulted
in the rapid resumption of food intake and a gradual gain of body
weight, which correlated with the gradual return of hyperinsulinemia
and insulin resistance. These results not only demonstrated that the
obese and diabetic phenotypes in the adult
ob
/
ob
mice are corrected by leptin gene
treatment but also provide confirming evidence that body weight control
may be critical in the long-term management of non-insulin-dependent
diabetes mellitus in obese patients.
Spectral diffusion and the energy landscape of a protein
Authors: Fritsch K. , Friedrich J. , Parak F. , Skinner J. L.
Published: July 2002

We present a novel type of spectral diffusion experiment in the
millikelvin range to characterize the energy landscape of a protein as
compared with that of a glass. We measure the time evolution of
spectral holes for more than 300 hr after well-defined initial
nonequilibrium conditions. We show that the model of noninteracting
two-level systems can describe spectral diffusion in the glass, but
fails for the protein. Our results further demonstrate that randomness
in the energy landscape of a protein shows features of organization.
There are “deep minimum” states separated by barriers, the
heights of which we are able to estimate. The energy landscape of a
glass is featureless by comparison.
Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.
Authors: Kamikubo H , Kataoka M , Váró G , Oka T , Tokunaga F , Needleman R , Lanyi J K
Published: July 2002

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees Celsius. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.
Cationic facial amphiphiles: a promising class of transfection agents.
Authors: Walker S , Sofia M J , Kakarla R , Kogan N A , Wierichs L , Longley C B , Bruker K , Axelrod H R , Midha S , Babu S , Kahne D
Published: July 2002

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.
Isolation of a Pax-6 homolog from the ribbonworm Lineus sanguineus.
Authors: Loosli F , Kmita-Cunisse M , Gehring W J
Published: July 2002

The Pax-6 genes of vertebrates and Drosophila encode transcription factors with highly conserved paired- and homeodomains. They are expressed in the nervous system and the developing eyes. Loss-of-function mutations in mammals and flies lead to a reduction or absence of the eyes. By ectopic expression of Pax-6 in Drosophila ectopic eyes can be induced, indicating a determinative role in eye morphogenesis. We have isolated a Pax-6 homolog of the ribbonworm Lineus sanguineus. This gene shares extensive sequence identity and several conserved splice sites with the mammalian and Drosophila genes. During head regeneration the L. sanguineus Pax-6 homolog is expressed in the central nervous system, in the cerebral organ, and in the eye region. These findings support the hypothesis that Pax-6 was present in primitive metazoa before the evolutionary separation of vertebrates and arthropods and suggest a fundamental role in eye and central nervous system development.
Successful expression of human factor IX following repeat administration of adenoviral vector in mice.
Authors: Walter J , You Q , Hagstrom J N , Sands M , High K A
Published: July 2002

Adenoviral vectors can direct high-level expression of a transgene, but, due to a host immune response to adenoviral antigens, expression is of limited duration, and repetitive administration has generally been unsuccessful. Exposure to foreign proteins beginning in the neonatal period may alter or ablate the immune response. We injected adult and neonatal (immunocompetent) CD-1 mice intravenously with an adenoviral vector expressing human blood coagulation factor IX. In both groups of mice, expression of human factor IX persisted for 12-16 weeks. However, in mice initially injected as adults, repeat administration of the vector resulted in no detectable expression of the transgene, whereas in mice initially injected in the neonatal period, repeat administration resulted in high-level expression of human factor IX. We show that animals that fail to express the transgene on repeat administration have developed high-titer neutralizing antibodies to adenovirus, whereas those that do express factor IX have not. This experimental model suggests that newborn mice can be tolerized to adenoviral vectors and demonstrates that at least one repeat injection of the adenoviral vector is possible; the model will be useful in elucidating the immunologic mechanisms underlying successful repeat administration of adenoviral vectors.
Voltage gating of
Escherichia coli
porin channels: Role of the constriction loop
Authors: Phale Prashant S. , Schirmer Tilman , Prilipov Alexej , Lou Kuo-Long , Hardmeyer Ariane , Rosenbusch Jurg P.
Published: July 2002

In the homotrimeric OmpF porin from
Escherichia coli
, each channel is constricted by a loop protruding into the β-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Δ116–120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.
Glycine-induced long-term potentiation is associated with structural and functional modifications of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors
Authors: Musleh Wael , Bi Xiaoning , Tocco Georges , Yaghoubi Shahriar , Baudry Michel
Published: July 2002

Global long-term potentiation (LTP) was induced in organotypic hippocampal slice cultures by a brief application of 10 mM glycine. Glycine-induced LTP was occluded by previous theta burst stimulation-induced potentiation, indicating that both phenomena share similar cellular processes. Glycine-induced LTP was associated with increased [
3
H]α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) binding in membrane fractions as well as increased amount of a selective spectrin breakdown product generated by calpain-mediated spectrin proteolysis. Antibodies against the C-terminal (C-Ab) and N-terminal (N-Ab) domains of GluR1 subunits were used to evaluate structural changes in AMPA receptor properties resulting from glycine-induced LTP. No quantitative or qualitative changes were observed in Western blots from membrane fractions prepared from glycine-treated slices with C-Ab. In contrast, Western blots stained with N-Ab revealed the formation of a 98-kDa species of GluR1 subunits as well as an increased amount of immunoreactivity after glycine-induced LTP. The amount of spectrin breakdown product was positively correlated with the amount of the 98-kDa species of GluR1 after glycine treatment. Functional modifications of AMPA receptors were evaluated by determining changes in the effect of pressure-applied AMPA on synaptic responses before and after glycine-induced LTP. Glycine treatment produced a significant increase in AMPA receptor function after potentiation that correlated with the degree of potentiation. The results indicate that LTP induction produces calpain activation, truncation of the C-Ab domain of GluR1 subunits of AMPA receptors, and increased AMPA receptor function. They also suggest that insertion of new receptors takes place after LTP induction.
Fos
B mutant mice: Loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to cocaine’s psychomotor and rewarding effects
Authors: Hiroi Noboru , Brown Jennifer R. , Haile Colin N. , Ye Hong , Greenberg Michael E. , Nestler Eric J.
Published: July 2002

Chronic exposure to cocaine leads to prominent, long-lasting changes in behavior that characterize a state of addiction. The striatum, including the nucleus accumbens and caudoputamen, is an important substrate for these actions. We previously have shown that long-lasting Fos-related proteins of 35–37 kDa are induced in the striatum by chronic cocaine administration. In the present study, the identity and functional role of these Fos-related proteins were examined using
fos
B mutant mice. The striatum of these mice completely lacked basal levels of the 35- to 37-kDa Fos-related proteins as well as their induction by chronic cocaine administration. This deficiency was associated with enhanced behavioral responses to cocaine:
fos
B mutant mice showed exaggerated locomotor activation in response to initial cocaine exposures as well as robust conditioned place preference to a lower dose of cocaine, compared with wild-type littermates. These results establish the long-lasting Fos-related proteins as products of the
fos
B gene (specifically ΔFosB isoforms) and suggest that transcriptional regulation by
fos
B gene products plays a critical role in cocaine-induced behavioral responses. This finding demonstrates that a Fos family member protein plays a functional role in behavioral responses to drugs of abuse and implicates
fos
B gene products as important determinants of cocaine abuse.
Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway
Authors: Pumiglia Kevin M. , Decker Stuart J.
Published: July 2002

The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation. The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined. Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF. PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21
Cip1/WAF1
. To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (ΔRAF-1:ER) was expressed in these cells. Activation of ΔRAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21
Cip1/WAF1
and inhibition of CDK2 activity. These effects of ΔRAF-1:ER activation were all reversed by treatment of cells with PD98059. These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest.
Activation of human B-MYB by cyclins
Authors: Sala Arturo , Kundu Mondira , Casella Ida , Engelhard Andrew , Calabretta Bruno , Grasso Luigi , Paggi Marco G. , Giordano Antonio , Watson Roger J. , Khalili Kamel , Peschle Cesare
Published: July 2002

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human
B-myb
gene can be activated
in vivo
by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the
cdc2
promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an
in vitro
transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.
Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein
Authors: Dey Saibal , Ramachandra Muralidhara , Pastan Ira , Gottesman Michael M. , Ambudkar Suresh V.
Published: July 2002

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [
125
I]iodoarylazidoprazosin ([
125
I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [
125
I]IAAP binding to the N- and C-terminal halves.
cis
(
Z
)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [
125
I]IAAP for the C-terminal half of the protein (C-site) by reducing the
K
d
from 20 to 6 nM without changing the labeling or affinity (
K
d
= 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [
125
I]IAAP binding to the C-site was increased 5- to 6-fold by
cis
(
Z
)-flupentixol without any effect on the N-site. In addition, [
125
I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.
Fluorescence and photobleaching dynamics of single light-harvesting complexes
Authors: Bopp Martin A. , Jia Yiwei , Li Liangquan , Cogdell Richard J. , Hochstrasser Robin M.
Published: July 2002

Single light-harvesting complexes LH-2 from
Rhodopseudomonas acidophila
were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 ± 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only ≈3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of ≈0.1 s. About 490 molecules were examined.
Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations
Authors: Antoniou Dimitri , Schwartz Steven D.
Published: July 2002

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989)
Science
243, 1325–1330; Grant, K. L. & Klinman, J. P. (1989)
Biochemistry
28, 6597–6605; and Bahnson, B. J. & Klinman, J. P. (1995)
Methods Enzymol.
249, 373–397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991)
J. Chem. Phys.
94, 3619–3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.
Syntax processing by auditory cortical neurons in the FM–FM area of the mustached batPteronotus parnellii
Authors: Esser Karl-Heinz , Condon Curtis J. , Suga Nobuo , Kanwal Jagmeet S.
Published: July 2002

Syntax denotes a rule system that allows one to predict the sequencing of communication signals. Despite its significance for both human speech processing and animal acoustic communication, the representation of syntactic structure in the mammalian brain has not been studied electrophysiologically at the single-unit level. In the search for a neuronal correlate for syntax, we used playback of natural and temporally destructured complex species-specific communication calls—so-called composites—while recording extracellularly from neurons in a physiologically well defined area (the FM–FM area) of the mustached bat’s auditory cortex. Even though this area is known to be involved in the processing of target distance information for echolocation, we found that units in the FM–FM area were highly responsive to composites. The finding that neuronal responses were strongly affected by manipulation in the time domain of the natural composite structure lends support to the hypothesis that syntax processing in mammals occurs at least at the level of the nonprimary auditory cortex.
Spatio-temporally controlled site-specific somatic mutagenesis in the mouse
Authors: Brocard Jacques , Warot Xavier , Wendling Olivia , Messaddeq Nadia , Vonesch Jean-Luc , Chambon Pierre , Metzger Daniel
Published: July 2002

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination–excision between two
loxP
sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ER
T
), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ER
T
under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (≈40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER
T
in a given cell type, we have now crossed Cre-ER
T
-expressing mice with reporter mice in which expression of
Escherichia coli
β-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER
T
. These results indicate that cell-specific expression of Cre-ER
T
in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing
loxP
sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.