bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the prostate for destruction through a vascular address
Authors: Arap Wadih , Haedicke Wolfgang , Bernasconi Michele , Kain Renate , Rajotte Daniel , Krajewski Stanislaw , Ellerby H. Michael , Bredesen Dale E. , Pasqualini Renata , Ruoslahti Erkki
Published: July 2002

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10–15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk.
Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature
Authors: Essler Markus , Ruoslahti Erkki
Published: July 2002

In vivo
phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the
in vitro
binding of the phage-displayed cDNA to the peptide and the
in vivo
homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.
A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.
Authors: Wang Y , Mangel S C
Published: July 2002

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.
Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera.
Authors: Htun H , Barsony J , Renyi I , Gould D L , Hager G L
Published: July 2002

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.
Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.
Authors: Bugge T H , Xiao Q , Kombrinck K W , Flick M J , Holmbäck K , Danton M J , Colbert M C , Witte D P , Fujikawa K , Davie E W , Degen J L
Published: July 2002

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.
Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.
Authors: Levi E , Fridman R , Miao H Q , Ma Y S , Yayon A , Vlodavsky I
Published: July 2002

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.
DNA bending by hexamethylene-tethered ammonium ions.
Authors: Strauss J K , Roberts C , Nelson M G , Switzer C , Maher L J
Published: July 2002

DNA is bent when complexed with certain proteins. We are exploring the hypothesis that asymmetric neutralization of phosphate charges will cause the DNA double helix to collapse toward the neutralized face. We have previously shown that DNA spontaneously bends toward one face of the double helix when it is partially substituted with neutral methylphosphonate linkages. We have now synthesized DNA duplexes in which cations are tethered by hexamethylene chains near specific phosphates. Electrophoretic phasing experiments demonstrate that tethering six ammonium ions on one helical face causes DNA to bend by approximately 5 degrees toward that face, in qualitative agreement with predictions. Ion pairing between tethered cations and DNA phosphates provides a new model for simulating the electrostatic consequences of phosphate neutralization by proteins.
In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections.
Authors: Wu X R , Sun T T , Medina J J
Published: July 2002

Urinary tract infections, caused mainly by Escherichia coli, are among the most common infectious diseases. Most isolates of the uropathogenic E.coli can express type 1 and P fimbriae containing adhesins that recognize cell receptors. While P fimbriae recognize kidney glycolipid receptors and are involved in peyelonephritis, the urothelial for type 1 fimbriae were not identified. We show that type 1-fimbriated E. coli recognize uroplakins Ia and Ib, two major glycoproteins of urothelial apical plaques. Anchorage of E. coli to urothelial surface via type 1 fimbriae-uroplakin I interactions may play a role in its bladder colonization and eventual ascent through the ureters, against urine flow, to invade the kidneys.
Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.
Authors: Ossovskaya V S , Mazo I A , Chernov M V , Chernova O B , Strezoska Z , Kondratov R , Stark G R , Chumakov P M , Gudkov A V
Published: July 2002

p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.
Gain and kinetics of activation in the G-protein cascade of phototransduction.
Authors: Lamb T D
Published: July 2002

The guanine nucleotide binding protein (G protein) cascade underlying phototransduction is one of the best understood of all signaling pathways. The diffusional interactions of the proteins underlying the cascade have been analyzed, both at a macroscopic level and also in terms of the stochastic nature of the molecular contacts. In response to a single activated rhodopsin (R*) formed as a result of a single photon hit, it can be shown that molecules of the G-protein transducin will be activated approximately linearly with time. This, in turn, will cause the number of activated molecules of the effector protein (the phosphodiesterase) also to increase linearly with time. These kinetics of protein activation provide an accurate description of the time course of the rising phase of the photoreceptor's electrical response over a wide range of flash intensities. Recent estimates indicate that at room temperature each R* triggers activation of the phosphodiesterase at a rate of 1000-2000 subunits.s-1. Now that a quantitative description of the activation steps in transduction has been obtained, perhaps the greatest challenge for the future is to provide a comprehensive description of the shutoff reactions, so that a complete account of the photoreceptor's response to light can be achieved.
A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.
Authors: Goto K , Kondo H
Published: July 2002

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.
An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.
Authors: Jamil H , Gordon D A , Eustice D C , Brooks C M , Dickson J K , Chen Y , Ricci B , Chu C H , Harrity T W , Ciosek C P , Biller S A , Gregg R E , Wetterau J R
Published: July 2002

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.
Nature knows best: An amazing reaction cascade is uncovered by design and discovery
Authors: Heathcock Clayton H.
Published: July 2002

The
Daphniphyllum
alkaloids are a group of highly
complex polycyclic alkaloids. Examination of the structures if several
members of this family of natural products led to a hypothesis about
their mode of biosynthesis (depicted in Scheme
SI
). Based on this
hypothetical biosynthetic pathway, a laboratory synthesis was designed
that incorporated as a key transformation the novel one-pot
transformation of dialdehyde 24 to pentacyclic unsaturated amine 25.
This process turned out to be an exceptionally efficient way to
construct the pentacyclic nucleus of the
Daphniphyllum
alkaloids. However, a purely fortuitous discovery, resulting from
accidental use of methylamine rather than ammonia, led to a great
improvement in the synthesis and suggests an even more attractive
possible biosynthesis.
Scanning mutagenesis of the putative transmembrane segments of K
ir
2.1, an inward rectifier potassium channel
Authors: Collins Anthony , Chuang Huai-hu , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

Structural models of inward rectifier K
+
channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in K
ir
2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in
Xenopus
oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.
The epitopes for natural polyreactive antibodies are rich in proline
Authors: Tchernychev Boris , Cabilly Shmuel , Wilchek Meir
Published: July 2002

“Natural” polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor α) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common “public” epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.
Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries
Authors: Uebel Stephan , Kraas Wolfgang , Kienle Stefan , Wiesmüller Karl-Heinz , Jung Günther , Tampé Robert
Published: July 2002

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containingdamino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.
Molecular switch in signal transduction: Reaction paths of the conformational changes in
ras
p21
Authors: Ma Jianpeng , Karplus Martin
Published: July 2002

Conformational changes in
ras
p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.
Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin
Authors: Wu Wei , Bancroft John D. , Suttie J. W.
Published: July 2002

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FII
RHH
and FII
HRR
, FII
RRH
and FII
HHR
, FII
RHR
and FII
HRH
) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FII
HrhH
and FII
HhrH
, FII
RrhR
, and FII
RhrR
) was used to determine that the first of the two kringles plays a more important role in the recognition process.
Ambient UV-B radiation causes deformities in amphibian embryos
Authors: Blaustein Andrew R. , Kiesecker Joseph M. , Chivers Douglas P. , Anthony Robert G.
Published: July 2002

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual’s subsequent survival after hatching.
N
-methyl-
d
-aspartate receptor activation and visual activity induce elongation factor-2 phosphorylation in amphibian tecta: A role for
N
-methyl-
d
-aspartate receptors in controlling protein synthesis
Authors: Scheetz A. J. , Nairn Angus C. , Constantine-Paton Martha
Published: July 2002

N
-methyl-
d
-aspartate receptor (NMDAR) activation has been implicated in forms of synaptic plasticity involving long-term changes in neuronal structure, function, or protein expression. Transcriptional alterations have been correlated with NMDAR-mediated synaptic plasticity, but the problem of rapidly targeting new proteins to particular synapses is unsolved. One potential solution is synapse-specific protein translation, which is suggested by dendritic localization of numerous transcripts and subsynaptic polyribosomes. We report here a mechanism by which NMDAR activation at synapses may control this protein synthetic machinery. In intact tadpole tecta, NMDAR activation leads to phosphorylation of a subset of proteins, one of which we now identify as the eukaryotic translation elongation factor 2 (eEF2). Phosphorylation of eEF2 halts protein synthesis and may prepare cells to translate a new set of mRNAs. We show that NMDAR activation-induced eEF2 phosphorylation is widespread in tadpole tecta. In contrast, in adult tecta, where synaptic plasticity is reduced, this phosphorylation is restricted to short dendritic regions that process binocular information. Biochemical and anatomical evidence shows that this NMDAR activation-induced eEF2 phosphorylation is localized to subsynaptic sites. Moreover, eEF2 phosphorylation is induced by visual stimulation, and NMDAR blockade before stimulation eliminates this effect. Thus, NMDAR activation, which is known to mediate synaptic changes in the developing frog, could produce local postsynaptic alterations in protein synthesis by inducing eEF2 phosphorylation.
A binding site for Pax proteins regulates expression of the gene for the neural cell adhesion molecule in the embryonic spinal cord
Authors: Holst Brent D. , Wang Yibin , Jones Frederick S. , Edelman Gerald M.
Published: July 2002

The neural cell adhesion molecule (N-CAM) mediates cell–cell interactions and is expressed in characteristic spatiotemporal patterns during development. In previous studies of factors that control N-CAM gene expression, we identified a binding site for the paired domain of Pax proteins (designated PBS) in the mouse N-CAM promoter. In this study, we demonstrate that a transcription factor known to be important for development of the central nervous system, Pax-6, binds to the N-CAM PBS and show that the PBS can influence N-CAM expression
in vivo
. Pax-6, produced in COS-1 cells, bound to the PBS through two half-sites, PBS-1 and PBS-2; mutations in both of these sites completely disrupted binding. Moreover, nuclear extracts from embryonic day (E) 11.5 mouse embryos bound to the PBS, and this binding was inhibited by antibodies to Pax-6. To determine the role of the PBS
in vivo
, we generated transgenic mice with N-CAM promoter/
lacZ
gene constructs containing either a wild-type or a mutated PBS. Mutations in PBS-1 and PBS-2 decreased the extent of β-galactosidase expression in the mantle layer of the spinal cord limiting it to ventral regions at E11.5. At E14.5, these mutations eliminated most of the expression that was seen in the wild-type spinal cord. Taken together with our previous observations that the PBS binds multiple Pax proteins, the data indicate that such binding contributes to the regulation of N-CAM gene expression during neural development.
Receptor and Gβγ isoform-specific interactions with G protein-coupled receptor kinases
Authors: Daaka Yehia , Pitcher Julie A. , Richardson Mark , Stoffel Robert H. , Robishaw Janet D. , Lefkowitz Robert J.
Published: July 2002

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous
in vitro
studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free βγ subunits of heterotrimeric G proteins (Gβγ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβγ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβγ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ
1
γ
2
, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβγ complex. That Gβγ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβγ complex is formed after agonist occupancy of the lysophosphatidic acid and β-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβγ complex after stimulation of all three GPCRs. This Gβγ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ
1
and Gβ
2
but not Gβ
3
, while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβγ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.
Molecular structure of a wheat chromosome end healed after gametocidal gene-induced breakage
Authors: Tsujimoto H. , Yamada T. , Sasakuma T.
Published: July 2002

In the progeny of the monosomic addition line of common wheat,
Triticum aestivum
, carrying the gametocidal chromosome of
Aegilops cylindrica
, deletion chromosomes carrying the break point within the nucleolar organizing region of chromosome 1B appeared. Attempts were made to amplify the break points by PCR using primers of telomere and rDNA (rRNA gene). In one deletion line, specific amplification of DNA fragments including the 18S rRNA gene, telomere repeats, and their junction occurred. At the junction of telomere and rRNA gene there was a 31-bp inverted duplication of the rRNA gene. Telomere sequences were initiated from the sequence TAG in the duplication. Between the duplications a small sequence was also inserted. This novel DNA sequence at the break point indicates that the breakage–fusion–bridge cycle(s) took place after the first chromatin breakage by the gametocidal gene.
Development of medullary thyroid carcinoma in transgenic mice expressing the
RET
protooncogene altered by a multiple endocrine neoplasia type 2A mutation
Authors: Michiels Francine-Marie , Chappuis Sophie , Caillou Bernard , Pasini Andrea , Talbot Monique , Monier Roger , Lenoir Gilbert M. , Feunteun Jean , Billaud Marc
Published: July 2002

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome that comprises three clinical subtypes: MEN type 2A (MEN-2A), MEN type 2B (MEN-2B), and familial medullary thyroid carcinoma (FMTC). Medullary thyroid carcinoma (MTC), a malignant tumor arising from calcitonin-secreting thyroid C cells, is the cardinal disease feature of this syndrome, and mortality in affected MEN-2 patients is mainly caused by this malignancy. Germ-line mutations of the
RET
protooncogene, which encodes a receptor tyrosine kinase, are responsible for these three neoplastic-prone disorders.
MEN2
mutations convert the
RET
protooncogene in a dominantly acting oncogene as a consequence of the ligand-independent activation of the tyrosine kinase. The majority of
MEN2A
and
FMTC
mutations are located in the extracellular domain and cause the replacement of one of five juxtamembrane cysteines by a different amino acid. To examine whether expression of a
MEN2A
allele of
RET
results in transformation of C cells, we have used the transgenic approach. Expression of the
RET
gene altered by a
MEN2A
mutation was targeted in C cells by placing the transgene under the control of the calcitonin gene-related peptide/calcitonin promoter. Animals of three independent transgenic mouse lines, which expressed the transgene in the thyroid, displayed overt bilateral C cell hyperplasia as early as 3 weeks of age and subsequently developed multifocal and bilateral MTC. Moreover, these tumors were morphologically and biologically similar to human MTC which afflicts MEN- 2 individuals. These findings provide evidence that the
MEN2A
mutant form of
RET
is oncogenic in parafollicular C cells and suggest that these transgenic mice should prove a valuable animal model for hereditary MTC.
Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β-galactosidase in mouse embryos and hematopoietic cells
Authors: Zambrowicz Brian P. , Imamoto Akira , Fiering Steve , Herzenberg Leonard A. , Kerr William G. , Soriano Philippe
Published: July 2002

The ROSAβgeo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral βgeo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5′ ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in
Caenorhabditis elegans
.
The muscle of Lawrence in
Drosophila
: A case of repeated evolutionary loss
Authors: Gailey Donald A. , Ohshima Saiyou , Santiago Sabrina J.-M. , Montez Jason M. , Arellano André R. , Robillo Josephine , Villarimo Cynthia A. , Roberts Langdon , Fine Eric , Villella Adriana , Hall Jeffrey C.
Published: July 2002

The muscle of Lawrence (MOL) is a bilaterally symmetrical muscle spanning the tergite of the fifth abdominal segment of adult male
Drosophila melanogaster.
It is not, however, a general feature of male-specific development within the subfamily Drosophilinae. Of 95 species surveyed within this subfamily, 67 exist with no MOL at all. By drawing comparisons with published cladograms of species relatedness, three conclusions regarding the evolutionary history of the MOL are made: (
i
) The MOL predates the major radiations of the genus
Drosophila,
given its presence in earlier-branching
Chymomyza
and
Scaptodrosophila
; the MOL has been subsequently excluded in at least one present species of each of these two primitive genera. (
ii
) Within the genus
Drosophila
the MOL is present sporadically in the radiation of the subgenus
Sophophora
, showing repetitive loss even in very close evolutionary lineages. (
iii
) The MOL may have been entirely excluded from the prolific radiation of the subgenus
Drosophila
. Thus the MOL shows a uniquely incongruous pattern of presence or absence relative to accepted drosophilid phylogeny.
Mapping the σ
70
subunit contact sites on
Escherichia coli
RNA polymerase with a σ
70
-conjugated chemical protease
Authors: Owens Jeffrey T. , Miyake Reiko , Murakami Katsuhiko , Chmura Albert J. , Fujita Nobuyuki , Ishihama Akira , Meares Claude F.
Published: July 2002

The core enzyme of
Escherichia coli
RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ
70
, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α
2
ββ′), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(
p
-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ
70
by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ
70
was bound to the core enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996)
Proc. Natl. Acad. Sci. USA
93, 71–75], the cut sites from different probe-modified σ
70
proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).
Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI
Authors: Yang Xiaojin , Renken Remco , Hyder Fahmeed , Siddeek Mohamed , Greer Charles A. , Shepherd Gordon M. , Shulman Robert G.
Published: July 2002

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10
−2
dilution of saturated vapor) with spatial and temporal resolutions of 220×220×1,000 μm and 36 s. The laminar structure of the OB was clearly depicted by high-resolution
in vivo
anatomical MRI with spatial resolution of 110×110×1,000 μm. In repeated BOLD fMRI measurements, highly significant (
P
< 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (ΔS/S = 10–30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.
Reorganization of auditory cortex in tinnitus
Authors: Mühlnickel Werner , Elbert Thomas , Taub Edward , Flor Herta
Published: July 2002

Magnetic source imaging was used to determine whether tonotopy in auditory cortex of individuals with tinnitus diverges from normative functional organization. Ten tinnitus subjects and 15 healthy controls were exposed to four sets of tones while magnetoencephalographic recordings were obtained from the two cortical hemispheres in sequence. A marked shift of the cortical representation of the tinnitus frequency into an area adjacent to the expected tonotopic location was observed. The Euclidean distance of the tinnitus frequency from the trajectory of the tonotopic map was 5.3 mm (SD = 3.1) compared with a distance of 2.5 mm (SD = 1.3) of a corresponding frequency in the healthy controls (
t
= 3.13,
P
< 0.01). In addition, a strong positive correlation was found between the subjective strength of the tinnitus and the amount of cortical reorganization (
r
= 0.82,
P
< 0.01). These results demonstrate that tinnitus is related to plastic alterations in auditory cortex. Similarities between these data and the previous demonstrations that phantom limb pain is highly correlated with cortical reorganization suggest that tinnitus may be an auditory phantom phenomenon.
MEKK1/JNK signaling stabilizes and activates p53
Authors: Fuchs Serge Y. , Adler Victor , Pincus Matthew R. , Ronai Ze’ev
Published: July 2002

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH
2
kinase (JNK). As JNK association with p53 plays an important role in p53 stability, in the present study we have elucidated the relationship between the JNK-signaling pathway and p53 stability and activity. Expression of a constitutively active form of JNKK upstream kinase, mitogen-activated protein kinase kinase kinase (ΔMEKK1), increased the level of the exogenously transfected form of p53 in p53 null (10.1) cells as well as of endogenous p53 in MCF7 breast cancer cells. Increased p53 level by forced expression of ΔMEKK1 coincided with a decrease in p53 ubiquitination
in vivo
and with prolonged p53 half-life. Computerized modeling of the JNK-binding site (amino acids 97–116; p7 region) enabled us to design mutations of exposed residues within this region. Respective mutations (p53
101-5-8
) and deletion (p53
Δp7
) forms of p53 did not exhibit the same increase in p53 levels upon ΔMEKK1 expression.
In vitro
phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, ΔMEKK1 expression increased p53 phosphorylation by immunopurified JNK and dissociated p53–Mdm2 complexes. Transcriptional activity of p53, as measured via
mdm
2 promoter-driven luciferase, exhibited a substantial increase in ΔMEKK1-expressing cells. Cotransfection of p53 and ΔMEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effectors contribute to the ability of p53 to mediate programmed cell death. Our results point to the role of MEKK1-JNK signaling in p53 stability, transcriptional activities, and apoptotic capacity as part of the cellular response to stress.
Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa
Authors: Cui Xiangfeng , Li Honghua
Published: July 2002

To determine human Ig heavy chain variable region (VH) gene segment organization on individual homologous chromosomes, an efficient approach has been developed. Single spermatozoa were used as subjects for the study. Upon sperm lysis, VHregions in each sperm were randomly sheared into fragments by the random Brownian force. The fragments were separated from each other by aliquoting the lysate into a certain number of tubes. The gene segments in the VH1 and VH4 families in each tube were identified by denaturing gradient gel electrophoresis after PCR amplification. The polymorphic VHsequences were used to determine the parental origins of the analyzed sperm. VHsegment organization in the parental haplotypes was determined by aligning the overlapping fragments from the spermatozoa with the corresponding haplotypes. Based on this comparison between the resulting haplotype maps and the composite map reported previously, the VHregion on chromosome 14 could be subdivided into four portions. The numbers and compositions of the VHgene segments differ considerably among the maps in two portions, but are highly conserved in the other two. The data also indicate that the VHregion on chromosome 15 may contain a large duplicated block with copy number varying among haplotypes. The approach used in the present study may be used to construct high-resolution haplotype maps without molecular cloning.