bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone.
Authors: Smith C L , Conneely O M , O'Malley B W
Published: May 2006

It has been previously demonstrated that several members of the steroid receptor superfamily may be activated by the neurotransmitter dopamine in the apparent absence of cognate ligand. We have examined wild-type and mutant human estrogen receptors (ERs, [Gly400]ER and [Val400]ER, respectively) for their abilities to activate ER-dependent transcription of a transgene in a ligand-independent manner. In cells expressing the wild-type ER, dopamine was nearly as effective as 17 beta-estradiol at inducing the chloramphenicol acetyltransferase activity of the reporter gene in a dose-dependent manner; simultaneous addition of suboptimal concentrations of 17 beta-estradiol and dopamine stimulated transcription more than either compound alone. Dopamine alone was unable to induce gene expression in cells expressing [Val400]ER mutant receptors, but concomitant treatment with 17 beta-estradiol produced a synergistic increase in transcription, suggesting that the ligand may alter the mutant receptor's conformation such that it can be activated subsequently by a dopaminergic signaling mechanism. In the presence of the antiestrogen ICI 164,384, dopamine-stimulated gene expression was undetectable in cells expressing either form of ER. However, simultaneous treatment of cells expressing wild-type ER with trans-4-hydroxytamoxifen and dopamine resulted in transgene expression that was additive in nature compared to either compound alone; similar treatment of cells expressing [Val400]ER produced a synergistic increase. Our results suggest that ligand and ligand-independent activation of the ER initiate from distinct pathways and that the latter may occur in a variety of target tissues subject to modulation by receptor ligands.
Interleukin 1 signaling occurs exclusively via the type I receptor.
Authors: Sims J E , Gayle M A , Slack J L , Alderson M R , Bird T A , Giri J G , Colotta F , Re F , Mantovani A , Shanebeck K
Published: May 2006

Two receptors for the proinflammatory cytokine interleukin 1 (IL-1) have been cloned and characterized biochemically. While it has been well established that the type I (80-kDa) IL-1 receptor can mediate responses to IL-1, the function of the type II (60-kDa) IL-1 receptor has been unknown. In this manuscript we describe experiments designed to ask whether the type II receptor is capable of delivering a biological signal. We have examined two types of experimental situation: responses to IL-1 in cells which express predominantly the type II receptor, and responses to IL-1 which have been suggested previously in the literature to be mediated by type II receptors. In both situations we find that the responses instead are mediated via type I receptors. A blocking antibody against the type II receptor never inhibits, and in fact sometimes enhances, the responses. We conclude that a very small number of type I receptors is sufficient to mediate all of the actions of IL-1 which we have examined here and that the function of the type II receptor may not be to transduce signals.
Process formation in Sf9 cells induced by the expression of a microtubule-associated protein 2C-like construct.
Authors: LeClerc N , Kosik K S , Cowan N , Pienkowski T P , Baas P W
Published: May 2006

To understand the roles of various microtubule-associated proteins (MAPs) in the development of axons and dendrites, we have expressed individual neuronal MAPs in normally rounded Sf9 host cells. We previously reported that expression of tau protein in these cells results in the elaboration of long processes containing dense bundles of microtubules (MTs). These bundles generally terminate in the hillock region of the cell body, and almost all of the MTs within the bundles are oriented with their plus ends distal to the cell body. Here we report the expression of a construct that approximates the MAP2C sequence and also induces the elaboration of processes with dense bundles of predominantly plus-end-distal MTs. Whereas tau generally results in a single process, there is a significantly greater tendency for the MAP2C-like construct to induce multiple processes. In contrast to the tau processes, the MT bundle in these processes extends far into the cell body. This latter observation suggests that MAP2C and tau have different effects on MT assembly and/or transport events in the cell. Although both of these MAPs can organize MTs that are competent to participate in process formation, the detailed organization of MTs induced by each of the two constructs is distinctive, and these differences may be relevant to axonal and dendritic differentiation.
The circadian oscillator is regulated by a very low fluence response of phytochrome in wheat.
Authors: Nagy F , Fejes E , Wehmeyer B , Dallman G , Schafer E
Published: May 2006

Expression of genes encoding the light-harvesting chlorophyll a/b binding proteins of photosystem II (Cab) in etiolated wheat seedlings is controlled by phytochrome and a circadian clock. Even photoconversion of <1% of phytochrome to its active form, which can be achieved by moonlight, induces the expression of the Cab genes, particularly that of the Cab-1 gene, in circadian fashion. Thus, this reaction shows the characteristics of a low and a very low fluence response. A single far-red light pulse given to an etiolated seedling is sufficient for a persistence of the circadian oscillation of the Cab-1 mRNA level for at least 100 h. Subsequent red (R) or long-wavelength far-red (RG9) light irradiations alter the free running rhythm. These observations indicate a change in sensitivity to phytochrome and/or a control by stable phytochrome. The latter hypothesis is supported by the observation that the level of Cab-1 mRNA is increased or decreased by a second R or RG9 light pulse, respectively.
Maize Spm transposable element has an enhancer-insensitive promoter.
Authors: Raina R , Cook D , Fedoroff N
Published: May 2006

We have used a transient assay system to investigate the promoter region of the maize Suppressor-mutator (Spm) transposable element. All of the sequence required for constitutive promoter activity is confined to the 0.2-kb sequence upstream from the transcription start site of the element at nt 209 and designated the upstream control region. The element's promoter is weak, lacks a conventional TATA box, and depends on the presence of multiple, short repetitive sequence elements. The Spm promoter is quite insensitive to the enhancer sequence of the cauliflower mosaic virus 35S promoter. Enhancer sensitivity can be restored by providing a -30 TATA sequence and removing the G + C-rich sequence encoding the untranslated leader of the element, designated the downstream control region. Although the downstream control region is without effect on Spm promoter activity, it completely inhibits the 35S core promoter and markedly inhibits activity of the complete 35S promoter. The properties of the Spm element's promoter buffer it from both mutational and position-dependent changes in activity. We suggest that the inherent characteristics of the promoter are part of the genetic mechanism that controls the element's transposition frequency, ensuring it remains low and insertion-site independent.
Transmutation of a heme protein.
Authors: Barker P D , Ferrer J C , Mylrajan M , Loehr T M , Feng R , Konishi Y , Funk W D , MacGillivray R T , Mauk A G
Published: May 2006

Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (E(m) = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group. Form BI has a covalently bound heme group coupled through a thioether linkage involving the beta carbon of the heme 4-vinyl group. Form BII is similar to BI except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (E(m) = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.
Binding and transcriptional activation of the promoter for the neural cell adhesion molecule by HoxC6 (Hox-3.3).
Authors: Jones F S , Holst B D , Minowa O , De Robertis E M , Edelman G M
Published: May 2006

Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.
Overexpression, characterization, and purification of a recombinant mouse immunophilin FKBP-52 and identification of an associated phosphoprotein.
Authors: Alnemri E S , Fernandes-Alnemri T , Nelki D S , Dudley K , DuBois G C , Litwack G
Published: May 2006

To gain insight into the structure and function of the immunophilin FKBP-52, a mouse FKBP-52 was overexpressed in Spodoptera frugiperda insect cells (Sf9 cells) with the baculovirus expression system. The purification and characterization of the recombinant FKBP-52 (rFKBP-52) was facilitated by incorporating a histidine 6-mer domain at its N terminus. The rFKBP-52 was highly purified on a N(i)2+ affinity resin with an estimated recovery of 10 mg of pure protein from 1 liter of Sf9 cell culture. Subcellular fractionation revealed that the rFKBP-52 is expressed predominantly in the nuclei of infected Sf9 cells maximally at 48 hr after infection, consistent with the nuclear localization of FKBP-52 in mammalian cells. The rFKBP-52 can be assembled in vitro with the glucocorticoid receptor complex, establishing its functionality and confirming that it is a component of the unactivated glucocorticoid receptor complex. The rFKBP-52 possesses an ATP/GTP binding activity that is stimulated by divalent cations. Furthermore, incubation of purified rFKBP-52 with [gamma-32P]ATP and MgCl2 resulted in the phosphorylation of a 59-kDa nuclear protein. Amino acid sequence analysis of this protein revealed that it is a phosphoprotein or kinase that is associated with the rFKBP-52.
Dominant lethality by expression of a catalytically inactive class I tRNA synthetase.
Authors: Schmidt E , Schimmel P
Published: May 2006

Alignment-guided mutagenesis was used to create an inactive, but toxic, aminoacyl-tRNA synthetase. An Asp-96-->Ala (D96A) replacement in the nucleotide binding fold of the class I Escherichia coli isoleucyl-tRNA synthetase inactivates the enzyme without disrupting its competence for binding isoleucine tRNA. Expression of plasmid-encoded mutant enzyme in a cell with a wild-type ileS chromosomal allele resulted in cell death. Introduction of a second K732T substitution previously shown to weaken tRNA binding gives an inactive D96A/K732T double mutant. Expression of the double mutant is not lethal to E. coli. D96A but not the double mutant significantly inhibited in vitro charging of isoleucine tRNA by the wild-type enzyme. The results suggest a dominant tRNA binding-dependent arrest of cell growth caused by a reduction in the pool of a specific tRNA. Specific tRNA binding drugs may have therapeutic applications for treatment of microbial pathogens.
Hammerhead ribozymes: importance of stem-loop II for activity.
Authors: Tuschl T , Eckstein F
Published: May 2006

The activity of several hammerhead ribozyme constructs with constant lengths of stems I and III of 5 nt each but with variously shortened stems II is reported. Stems with 2 bp rather than the conventional 4 bp show essentially unaltered catalytic activity, independent of the composition of the tetraloop. Further reduction in size to 1 bp or 0 bp decreases activity drastically. Inversion of the G10.1.C11.1 bp next to the invariant core leads to a loss in activity, even when the stem consists of 4 bp. Thus, the minimal structural requirement for stem-loop II is a 2-bp stem with a conserved G.C bp. The reduction in catalytic activity is predominantly a result of a decrease of catalytic constant kcat, whereas Km is only slightly affected. Thus, the structural requirement for optimal activity in these constructs where the chemical-cleavage step is rate limiting is determined by the stabilization of the transition state.
Reduced Mg2+ block of N-methyl-D-aspartate receptor-mediated synaptic potentials in developing visual cortex.
Authors: Kato N , Yoshimura H
Published: May 2006

Molecular cloning has demonstrated a diversity of artificially expressed N-methyl-D-aspartate (NMDA) receptors, implying a similar diversity of naturally occurring NMDA receptors in situ. Particularly significant was the success in expression of NMDA receptor classes exhibiting various sensitivities to Mg2+ block, a voltage-dependent channel blockade by Mg2+ that is essential to NMDA receptor functioning. Release from Mg2+ block often allows or facilitates the occurrence of long-term potentiation, a form of synaptic plasticity. Here we show that in the immature visual cortex, which is more susceptible to long-term potentiation than adult visual cortex, synaptically activated NMDA receptors, unlike those in the adult, have varying but clearly reduced sensitivities to Mg2+ block. We propose that the initially expressed, later-eliminated NMDA receptors exhibiting a reduced Mg2+ block may underlie the greater susceptibility to plasticity in the immature neocortex.
Early evolution of photosynthesis: clues from nitrogenase and chlorophyll iron proteins.
Authors: Burke D H , Hearst J E , Sidow A
Published: May 2006

Chlorophyll (Chl) is often viewed as having preceded bacteriochlorophyll (BChl) as the primary photoreceptor pigment in early photosynthetic systems because synthesis of Chl requires one fewer enzymatic reduction than does synthesis of BChl. We have conducted statistical DNA sequence analyses of the two reductases involved in Chl and BChl synthesis, protochlorophyllide reductase and chlorin reductase. Both are three-subunit enzymes in which each subunit from one reductase shares significant amino acid identity with a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The "chlorophyll iron protein" subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, also share amino acid sequence identity with the nitrogenase iron protein, encoded by nifH. When nitrogenase iron proteins are used as outgroups, the chlorophyll iron protein tree is rooted on the chlorine reductase lineage. This rooting suggests that the last common ancestor of all extant photosynthetic eubacteria contained BChl, not Chl, in its reaction center, and implies that Chl-containing reaction centers were a late invention unique to the cyanobacteria/chloroplast lineage.
Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.
Authors: Verma M , Davidson E A
Published: May 2006

To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.
L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons.
Authors: Dale N , Kandel E R
Published: May 2006

Although modulation of synaptic transmission between Aplysia mechanosensory and motor neurons has been an important model for processes thought to underlie simple forms of learning and memory, the nature of the fast excitatory transmitter utilized by the sensory neurons has remained obscure. To identify the sensory neuron transmitter, we first examined the detailed properties of the synaptic response evoked in motor neurons cocultured with pleural sensory neurons. The excitatory postsynaptic current had a nonlinear current-voltage relation with a reversal potential between 0 and 10 mV and a plateau region between -40 and -70 mV. When the concentration of Mg2+ in the artificial sea water was lowered to 5 mM, the current-voltage relation of the excitatory postsynaptic current became linear, suggesting that Mg2+ blocks the postsynaptic receptor in a voltage-dependent manner. After screening a variety of small molecules, we found that L-glutamate could mimic the actions of the sensory neuron transmitter: responses to L-glutamate also had a reversal potential between 0 and 10 mV and a nonlinear current-voltage relation that could be made linear by lowering external Mg2+. To demonstrate further similarity of action between L-glutamate and the endogenous transmitter, we utilized four antagonists (kynurenate, 6,7-dinitroquinoxaline-2,3-dione, D-aspartate, and D-glutamate) to block in a dose-dependent manner the actions of L-glutamate and the natural transmitter. We therefore suggest that the sensory neurons use a glutamate-like transmitter and favor L-glutamate itself, because no other naturally occurring amino acid that we have studied has had similar actions. As the postsynaptic receptor for the sensory neuron transmitter is weakly blocked in a voltage-dependent manner by Mg2+, the excitatory receptors innervated by the Aplysia sensory neuron may represent a distant precursor of the vertebrate N-methyl-D-aspartate receptor.
Isolation of x-ray-inducible transcripts from radioresistant human melanoma cells.
Authors: Boothman D A , Meyers M , Fukunaga N , Lee S W
Published: May 2006

Twelve x-ray-induced transcripts (xips), differentially expressed 8- to 230-fold in x-irradiated versus unirradiated radioresistant human melanoma (U1-Mel) cells, were isolated as cDNA clones (xip1 through xip12) after four rounds of differential hybridization. Northern analyses revealed rare, medium, and abundant xips, ranging in size from 1.2 to 10 kb. All transcripts were transiently expressed and induced by low, but not by high (> 600 cGy), doses of radiation. Three transcripts (xip4, -7, and -12) were induced only by ionizing radiation, and many (i.e., xip1, -2, -3, -5, -6, -8, -9, -10, and -11) were also induced by UV irradiation or phorbol 12-myristate 13-acetate. Heat shock did not induce any of the xips, but it decreased basal levels of xip4, -7, -11, and -12. Three xip cDNA clones were identified as encoding thymidine kinase, DT diaphorase, and tissue-type plasminogen activator. The remaining nine cDNA clones showed little homology to known genes. Three clones contained regions homologous to c-fes/fps protooncogene, recombination activating gene 1, or the human angiogenesis factor gene. X-ray-inducible genes may function in damaged cells to regulate DNA repair, apoptosis, mutagenesis, and carcinogenesis.
Human foamy virus genome possesses an internal, Bel-1-dependent and functional promoter.
Authors: Löchelt M , Muranyi W , Flügel R M
Published: May 2006

The human foamy or spumaretrovirus (HSRV) is a complex retrovirus that encodes the three retroviral genes gag, pol, and env and, in addition, at least three bel genes. The HSRV Bel-1 protein was identified as a transcriptional trans-activator. HSRV transcription starts in the 5' long terminal repeat at a defined guanine residue. We report here that a second efficiently utilized start site of transcription is contained within a HSRV env DNA sequence upstream of the bel genes. Bel-specific transcripts that initiate at the internal transcriptional start site at nt 9196 were identified in HSRV-infected cells by primer extension and S1 nuclease analysis, and the intragenic promoter was shown to be constitutively activated by Bel-1 in the HSRV provirus. In transient expression assays with indicator gene constructs, expression by the HSRV intragenic promoter/enhancer is Bel-1 dependent. The data provide evidence for an intragenic start site of transcription in the genome of a complex, exogenous human retrovirus and are discussed in terms of a model for regulating spumaretroviral gene expression.
Detection of recombinations between c-myc and immunoglobulin switch alpha in murine plasma cell tumors and preneoplastic lesions by polymerase chain reaction.
Authors: Janz S , Müller J , Shaughnessy J , Potter M
Published: May 2006

Virtually all murine plasmacytomas carry chromosomal translocations that activate c-myc. The predominant (approximately 90%) c-myc-activating chromosomal translocation in pristane (2,6,10,14-tetramethylpentadecane)-induced plasmacytomas in BALB/c mice is a reciprocal translocation t(12;15) in which an immunoglobulin heavy-chain switch sequence is joined to the 5' region of c-myc. The most common switch region involved is S alpha. We developed a direct PCR method to screen for recombinations between c-myc and S alpha. The critical step in establishing the method was the cloning and sequencing of the 5' flank of C alpha, a region with a reduced number of switch repeats that is much more favorable for designing specific PCR primers than the highly repetitive S alpha region. In applying this PCR method, we detected translocation-specific junction fragments in transplanted (10/16, 63%) and primary (5/15, 33%) plasmacytomas. Moreover, the sensitivity of a nested version of that technique allowed us to discern rare t(12;15)s in BALB/c mice in the preneoplastic stage of plasmacytoma-genesis (8/20 mice, 40%) as early as 30 days after administration of pristane. We conclude that t(12;15) is the probable primary, if not initiating, oncogenic step in plasmacytomagenesis.
A closed curve is much more than an incomplete one: effect of closure in figure-ground segmentation.
Authors: Kovács I , Julesz B
Published: May 2006

Detection of fragmented closed contours against a cluttered background occurs much beyond the local coherence distance (maximal separation between segments) of nonclosed contours. This implies that the extent of interaction between locally connected detectors is boosted according to the global stimulus structure. We further show that detection of a target probe is facilitated when the probe is positioned inside a closed circle. To explain the striking contour segregation ability found here, and performance enhancement inside closed boundaries, we propose the existence of a synergetic process in early vision.
Triplet structure of von Willebrand factor reflects proteolytic degradation of high molecular weight multimers.
Authors: Furlan M , Robles R , Affolter D , Meyer D , Baillod P , Lämmle B
Published: May 2006

High molecular weight (HMW) and low molecular weight (LMW) forms of von Willebrand factor (vWF) were isolated from normal human plasma in the presence of protease inhibitors. HMW and LMW vWF preparations were subjected to reduction of interdimeric disulfide bridges under mild reducing conditions. Following sodium dodecyl sulfate electrophoresis in 3% agarose, the vWF bands were detected by immunoblotting with a polyclonal rabbit anti-vWF antiserum as well as with two monoclonal antibodies directed against epitopes located in the NH2-terminal (MAb 418) or in the COOH-terminal (MAb 9) region of the vWF subunit. Our results suggest that the slowest migrating band of the dimeric triplet set of LMW vWF represents an asymmetric structure composed of an intact subunit to which one NH2-terminal and one COOH-terminal fragment are linked by disulfide bridges. The intermediate band of the first triplet of LMW vWF strongly reacted with MAb 9 but not with MAb 418, indicating that it represents a dimer of COOH-terminal fragments. The fastest migrating band of the same triplet is apparently a dimer of the NH2-terminal fragments because it reacted with MAb 418 but not with MAb 9. Each next higher family of triplets seems to contain one more asymmetric fragment of dimeric size. These results are compatible with a model according to which LMW forms of vWF are derived from HMW vWF by proteolytic cleavage in the circulating blood.
Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium.
Authors: Quinn T P , Peters K G , De Vries C , Ferrara N , Williams L T
Published: May 2006

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.
Local alterations of Krox-20 and Hox gene expression in the hindbrain suggest lack of rhombomeres 4 and 5 in homozygote null Hoxa-1 (Hox-1.6) mutant embryos.
Authors: Dollé P , Lufkin T , Krumlauf R , Mark M , Duboule D , Chambon P
Published: May 2006

It is unknown whether cross-regulatory interactions between homeotic genes, which have been shown to play an important role in the maintenance of their expression domains during Drosophila development, are also important during mammalian development. We have analyzed here the expression of Hox genes in Hoxa-1 (Hox-1.6) null mutant embryos to investigate the possible existence of regulatory interactions between Hoxa-1 and other Hox genes. We show that the absence of a functional Hoxa-1 gene product does not globally interfere with the expression of other Hox genes in terms of both spatial boundaries and transcript abundance. However, a limited area of the hindbrain shows a strong reduction in Hoxb-1 (Hox-2.9) and Krox-20 transcripts, which most likely reflects a marked reduction in size of the former fourth and fifth rhombomeres. These alterations coincide with the region that is subsequently affected in Hoxa-1 null mutant mice and suggest that the primary defects in this mutation are spatially restricted deletions of some rhombomeric structures.
Relevance of chemistry to conservation of isolated populations: the case of volatile leaf components of Dicerandra mints.
Authors: McCormick K D , Deyrup M A , Menges E S , Wallace S R , Meinwald J , Eisner T
Published: May 2006

Chemical analysis of the essential oils of four congeneric species of mint plant (Dicerandra spp.) endemic to Florida revealed a pattern of chemical similarity and dissimilarity that would not have been predicted on morphological or geographic criteria. Dicerandra christmanii differs fundamentally from its congeners in that it produces fewer compounds and lacks the acyclic components. Yet D. christmanii is more closely similar to Dicerandra frutescens than to other Dicerandra species in morphological characters and geographic range. We conclude that the potential chemical value of a species should not be prejudged on the basis of nonchemical characters and that designation of surplus plant populations by conservationists should be resisted unless such populations have also been chemically studied.
Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex.
Authors: Chitarra V , Alzari P M , Bentley G A , Bhat T N , Eiselé J L , Houdusse A , Lescar J , Souchon H , Poljak R J
Published: May 2006

Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.
Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen.
Authors: Havre P A , Gunther E J , Gasparro F P , Glazer P M
Published: May 2006

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.
Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.
Authors: Loo D T , Copani A , Pike C J , Whittemore E R , Walencewicz A J , Cotman C W
Published: May 2006

The molecular mechanism responsible for the neurodegeneration in Alzheimer disease is not known; however, accumulating evidence suggests that beta-amyloid peptide (A beta P) contributes to this degeneration. We now report that synthetic A beta Ps trigger the degeneration of cultured neurons through activation of an apoptotic pathway. Neurons treated with A beta Ps exhibit morphological and biochemical characteristics of apoptosis, including membrane blebbing, compaction of nuclear chromatin, and internucleosomal DNA fragmentation. Aurintricarboxylic acid, an inhibitor of nucleases, prevents DNA fragmentation and delays cell death. Our in vitro results suggest that apoptosis may play a role in the neuronal loss associated with Alzheimer disease.
Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.
Authors: Maryanoff B E , Qiu X , Padmanabhan K P , Tulinsky A , Almond H R , Andrade-Gordon P , Greco M N , Kauffman J A , Nicolaou K C , Liu A
Published: May 2006

The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.
Response variability in retinal ganglion cells of primates.
Authors: Croner L J , Purpura K , Kaplan E
Published: May 2006

The signal encoded by a sensory neuron is usually characterized as the cell's average response to repeated presentations of a stimulus. However, each stimulus presentation elicits a slightly different response. This response variability may obscure the signal represented by neural activity, but it might also be an important aspect of a neuron's message and in some instances may even serve useful function. Here we present evidence that response variability (noise) in primate retinal ganglion cells at photopic light levels is (i) independent of the amplitude of either the stimulus or the response and is therefore additive, (ii) independent of receptive field size and retinal eccentricity, and (iii) similar for all primate ganglion cells. Our results show that the primate retina maintains a uniform noise level across the entire visual field and suggest that the noise originates within the ganglion cells themselves.
Abundant mitochondrial DNA variation and world-wide population structure in humpback whales.
Authors: Baker C S , Perry A , Bannister J L , Weinrich M T , Abernethy R B , Calambokidis J , Lien J , Lambertsen R H , Ramírez J U , Vasquez O
Published: May 2006

Hunting during the last 200 years reduced many populations of mysticete whales to near extinction. To evaluate potential genetic bottlenecks in these exploited populations, we examined mitochondrial DNA control region sequences from 90 individual humpback whales (Megaptera novaeangliae) representing six subpopulations in three ocean basins. Comparisons of relative nucleotide and nucleotype diversity reveal an abundance of genetic variation in all but one of the oceanic subpopulations. Phylogenetic reconstruction of nucleotypes and analysis of maternal gene flow show that current genetic variation is not due to postexploitation migration between oceans but is a relic of past population variability. Calibration of the rate of control region evolution across three families of whales suggests that existing humpback whale lineages are of ancient origin. Preservation of preexploitation variation in humpback whales may be attributed to their long life-span and overlapping generations and to an effective, though perhaps not timely, international prohibition against hunting.
Delineation of an extended surface contact area on human CD4 involved in class II major histocompatibility complex binding.
Authors: Moebius U , Pallai P , Harrison S C , Reinherz E L
Published: May 2006

We describe a detailed mapping of the class II major histocompatibility complex (MHC) binding site using site-directed mutagenesis in conjunction with high-resolution CD4 structural data. Residues on all lateral surfaces of domain 1 and the neighboring portions of domain 2 participate in contacting class II MHC. Thus, in addition to the C'C" ridge that forms the human immunodeficiency virus type 1 gp120 binding site, apparent MHC contacts extend over the BED face of domain 1 and across the interdomain groove onto the FG loop of domain 2. Several models of the CD4/class II MHC interaction accounting for the extent of the CD4 surface involved are discussed, including the possibility that CD4 may contact more than one class II MHC molecule using different surfaces.
Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins.
Authors: Hickey M J , Hagen F S , Yagi M , Roth G J
Published: May 2006

Human platelet glycoprotein (GP) V (M(r) 83,300), whose primary structure is reported here, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome. With PCR techniques and platelet cDNA templates, 1.4 kb of GP V cDNA sequence was obtained that encodes 469 GP V amino acids. A genomic 3.5-kb BamHI fragment was then isolated that includes 3.46 kb of GP V cDNA sequence: the 1.7-kb open reading frame plus 2 bases of the 5' and 1.8 kb of the 3' untranslated regions. Northern blot analysis reveals three GP V platelet transcripts of 3.8, 4.2, and 5.2 kb. A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a "flank-LRG center-flank" array [Roth, G. J. (1991) Blood 77, 5-19] along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries.
Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction.
Authors: Silvennoinen O , Witthuhn B A , Quelle F W , Cleveland J L , Yi T , Ihle J N
Published: May 2006

Interleukin 3 (IL-3) regulates the proliferation and differentiation of hematopoietic cells. Although the IL-3 receptor chains lack kinase catalytic domains, IL-3 induces tyrosine phosphorylation of cellular proteins. To investigate the potential role of the JAK family of protein-tyrosine kinases in IL-3 signal transduction, we have obtained full-length cDNA clones for murine Jak1 and Jak2 protein-tyrosine kinases and prepared antiserum against the predicted proteins. Using antisera against Jak2, we demonstrate that IL-3 stimulation results in the rapid and specific tyrosine phosphorylation of Jak2 and activates its in vitro kinase activity.
Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria.
Authors: Welch M , Oosawa K , Aizawa S , Eisenbach M
Published: May 2006

Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three proteins (FliG, FliM, and FliN) and located at the base of the flagellar motor. The mechanism of function of the switch is unknown. Here we demonstrate that the diffusible clockwise-signal molecule, the CheY protein, binds to the switch, that the primary docking site is FliM, that the extent of CheY binding to FliM is dependent upon the phosphorylation level of CheY, and that it is unaffected by the other two switch proteins. This study provides a biochemical demonstration of binding of a signal molecule to the bacterial switch and demonstrates directly that phosphorylation regulates the activity of this molecule.
Transgenic mouse model of malignant skin melanoma.
Authors: Mintz B , Silvers W K
Published: May 2006

Tyr-SV40E transgenic mice are specifically susceptible to melanoma due to expression of the oncogene in pigment cells. Mice of the more susceptible lines die young of early-onset eye melanomas, when skin melanomas are still infrequent and benign. To surmount this obstacle, skin from donors of two high-susceptibility lines was grafted to Tyr-SV40E hosts of a low-susceptibility line of the same inbred strain, thereby enabling the skin to outlive the donors and continue to grow in immunocompetent but tolerant hosts. Unexpectedly, donor pigment cells in all the grafts soon selectively proliferated close to areas of greatest wound healing, forming a dense black tracery, especially at the outer rim of the grafts. These lesions slowly grew radially within the grafts, producing irregular greyish patches. Local vertical thickenings then appeared and developed into small melanomas, which soon ulcerated through the epidermis. The tumors rapidly enlarged and became deeply invasive. Discrete black nevi also arose, with many becoming larger and distinctly blue, but those not near areas of pronounced wound healing did not progress to malignancy. In this first series, malignant melanoma resulted in all the grafts from the more susceptible of two donor lines and in some grafts from the other line. Distant metastases occurred in some cases from each line. Most tumors were hypomelanotic and heterogeneous, with lobes or areas differing in melanization. The results strongly suggest that growth factors and cytokines--known to be produced in wound repair--are triggering the growth and malignant conversion of these genetically susceptible melanocytes and that in the graft situation we are merely witnessing a caricature--a usefully exaggerated manifestation of the true events underlying the genesis of melanomas. The striking resemblance to the human malignancy, the genetic uniformity and different susceptibilities of the transgenic lines, and the experimental possibilities in the grafted mice all make them an excellent model of the disease.
Physical association between the high-affinity IgG receptor (Fc gamma RI) and the gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma).
Authors: Scholl P R , Geha R S
Published: May 2006

To investigate the structural basis of transmembrane signaling via the high-affinity IgG receptor (Fc gamma RI), the identity of Fc gamma RI-associated proteins in THP-1 human monocytic cells was examined. Anti-Fc gamma RI monoclonal antibody (mAb) 197 immunoprecipitates from 125I-labeled THP-1 cells solubilized in 1% digitonin buffer were found to contain a protein migrating at 12 kDa on reduction. This protein comigrated with the 12-kDa protein precipitated by the anti-high-affinity IgE receptor gamma chain (Fc epsilon RI gamma) mAb 4D8. Similarly, a 70-kDa band immunoprecipitated by mAb 4D8 comigrated with the 70-kDa protein band corresponding to Fc gamma RI in mAb 197 immunoprecipitates. On two-dimensional nonreducing-reducing gel analysis, the 12-kDa protein present in both mAb 197 and mAb 4D8 immunoprecipitates migrated as a disulfide-linked homodimer. Analysis of 1% Nonidet P-40 eluates of the digitonin immunoprecipitates under reducing conditions demonstrated the presence of a 12-kDa band in the mAb 197 immunoprecipitate that could be reprecipitated with mAb 4D8. Conversely, a 70-kDa band in the mAb 4D8 immunoprecipitate could be reprecipitated with mAb 197. Similar to these findings, both mAb 197 and mAb 4D8 precipitated a 12-kDa disulfide-linked homodimeric protein from digitonin lysates of 125I-labeled human neutrophils after induction of Fc gamma RI expression with interferon gamma but not from unstimulated neutrophils. Northern blot analysis confirmed the presence of Fc epsilon RI gamma mRNA in interferon gamma-induced human neutrophils. We conclude that Fc epsilon RI gamma, a member of a family of proteins implicated in transmembrane signaling via immune recognition receptors, associates with Fc gamma RI in human cells.