bluuebunny/crossref_metadata_2025 huggingface dataset:
p21-activated kinase has substrate specificity similar to
Acanthamoeba
myosin I heavy chain kinase and activates
Acanthamoeba
myosin I
Authors: Brzeska Hanna , Knaus Ulla G. , Wang Zhen-Yuan , Bokoch Gary M. , Korn Edward D.
Published: July 2002

Acanthamoeba
class I myosins are unconventional, single-headed myosins that express actin-activated Mg
2+
-ATPase and
in vitro
motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of
Acanthamoeba
MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P
i
per mol) and activates
Acanthamoeba
myosin I as MIHCK does. These results, together with the known involvement of
Acanthamoeba
myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.
Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis
Authors: Bertin John , Armstrong Robert C. , Ottilie Sabine , Martin David A. , Wang Yu , Banks Sean , Wang Guang-Hua , Senkevich Tatiana G. , Alnemri Emad S. , Moss Bernard , Lenardo Michael J. , Tomaselli Kevin J. , Cohen Jeffrey I.
Published: July 2002

To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.
Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor–ligand interactions
Authors: Orlando Robert A. , Exner Markus , Czekay Ralf-Peter , Yamazaki Hajime , Saito Akihiko , Ullrich Robert , Kerjaschki Dontscho , Farquhar Marilyn Gist
Published: July 2002

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-β very low density lipoprotein (βVLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-βVLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111–1210. From these studies we conclude that the binding of apoE-βVLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.
The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins
Authors: Xu Pin , Mitchelhill Ken I. , Kobe Bostjan , Kemp Bruce E. , Zot Henry G.
Published: July 2002

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of
Acanthamoeba
myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3Δ977–994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of
Acanthamoeba
myosin-IC bound AD3p and not AD3Δ977–994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by
Caenorhabditis elegans
gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the
C. elegans
homologue define a novel family of bifunctional binding proteins.
Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation
Authors: Schmid Patricia C. , Paria B. C. , Krebsbach Randy J. , Schmid Harald H. O. , Dey S. K.
Published: July 2002

Anandamide (
N
-arachidonoylethanolamine) is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. This investigation demonstrates that the periimplantation mouse uterus contains the highest levels of anandamide (142–1345 pmol/μmol lipid P; 1–7 μg/g wet weight) yet discovered in a mammalian tissue. The levels fluctuate with the state of pregnancy; down-regulation of anandamide levels is associated with uterine receptivity, while up-regulation is correlated with uterine refractoriness to embryo implantation. Anandamide levels are highest during the nonreceptive phase in the pseudopregnant uterus and in the interimplantation sites, and lowest at the site of embryo implantation. The lower levels of uterine anandamide at the implantation sites may be a mechanism by which implanting embryos protect themselves from the detrimental effects of this endogenous ligand. We also observed a reduced rate of zona-hatching of blastocysts
in vitro
in the presence of anandamide, and inhibition of implantation by systemic administration of a synthetic cannabinoid agonist CP 55,940. These adverse effects were reversed by SR141716A, a specific CB1-R antagonist. Taken together, the results suggest that an aberrant synthesis of anandamide and/or expression of the cannabinoid receptors in the uterus/embryo may account for early pregnancy failure or female infertility.
Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor
Authors: Carpenter Laura Rocco , Farruggella Thomas J. , Symes Aviva , Karow Margaret L. , Yancopoulos George D. , Stahl Neil
Published: July 2002

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin’s action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr
986
within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr
986
to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin’s weight-reducing effects in obese individuals.
T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function
Authors: Löhning Max , Stroehmann Arne , Coyle Anthony J. , Grogan Jane L. , Lin Steven , Gutierrez-Ramos Jose-Carlos , Levinson Douglas , Radbruch Andreas , Kamradt Thomas
Published: July 2002

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-γ. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activatedin vivoorin vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 productionin vivofollowing adoptive transfer of Th2 cells.
Nedd4 mediates control of an epithelial Na
+
channel in salivary duct cells by cytosolic Na
+
Authors: Dinudom A. , Harvey K. F. , Komwatana P. , Young J. A. , Kumar S. , Cook D. I.
Published: July 2002

Epithelial Na
+
channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their α, β, and γ subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle’s syndrome) leads to increased Na
+
channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na
+
channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na
+
channel activity in response to increased intracellular Na
+
. We further show that Nedd4 operates downstream of G
o
in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na
+
channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na
+
channel activity when the Na
+
and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na
+
channels by intracellular Na
+
, and we suggest that the increased Na
+
channel activity observed in Liddle’s syndrome is attributable to the loss of this regulatory feedback system.
Hydration for a series of hydrocarbons
Authors: Mountain Raymond D. , Thirumalai D.
Published: July 2002

The hydrophobic hydration in a series of hydrocarbons is probed by using molecular dynamics simulations. The solutes considered range from methane to octane. Examination of the shapes of the hydration shell suggests that there is no single stable structure surrounding these solutes. The structure of the water molecules around the solute is not significantly perturbed, even for octane, and the hydrogen bond network is essentially preserved. The solutes are accommodated in the voids of the tetrahedral network of water in such a way as to leave the local environment almost intact. The hydrophobic hydration arises primarily because of the plasticity of the hydrogen bond network. Even for octane we find very little evidence for water-mediated interactions between nonbonded carbon atoms, leading us to suggest that the transition to globular conformations can only occur for very long, linear hydrocarbon chains.
Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk
Authors: Lee Kyung S. , Grenfell Tallessyn Z. , Yarm Frederic R. , Erikson Raymond L.
Published: July 2002

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homologue of
Saccharomyces cerevisiae
Cdc5. Here, we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a
cdc5–1
temperature-sensitive mutation and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.
Neural tube expression of pituitary adenylate cyclase-activating peptide (PACAP) and receptor: Potential role in patterning and neurogenesis
Authors: Waschek James A. , Casillas Robert A. , Nguyen Thinh B. , DiCicco-Bloom Emanuel M. , Carpenter Ellen M. , Rodriguez Williams I.
Published: July 2002

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by
in situ
hybridization that genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors (PAC
1
) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the
sonic hedgehog
- and PKA-dependent target gene
gli
-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.
Desoxyepothilone B: An efficacious microtubule-targeted antitumor agent with a promising
in vivo
profile relative to epothilone B
Authors: Chou Ting-Chao , Zhang Xiu-Guo , Balog Aaron , Su Dai-Shi , Meng Dongfang , Savin Kenneth , Bertino Joseph R. , Danishefsky Samuel J.
Published: July 2002

A new class of 16-membered macrolides, the epothilones (Epos), has been synthesized and evaluated for antitumor potential
in vitro
and
in vivo.
Recent studies in these and other laboratories showed that epothilones and paclitaxel (paclitaxel) share similar mechanisms of action in stabilizing microtubule arrays as indicated by binding-displacement studies, substitution for paclitaxel in paclitaxel-dependent cell growth, and electron microscopic examinations. The present study examined cell growth-inhibitory effects in two rodent and three human tumor cell lines and their drug-resistant sublines. Although paclitaxel showed as much as 1,970-fold cross-resistance to the sublines resistant to paclitaxel, adriamycin, vinblastine, or actinomycin D, most epothilones exhibit little or no cross-resistance. In multidrug-resistant CCRF-CEM/VBL
100
cells, IC
50
values for EpoA (1), EpoB (2), desoxyEpoA (3) (dEpoA), desoxyEpoB (4) (dEpoB), and paclitaxel were 0.02, 0.002, 0.012, 0.017, and 4.14 μM, respectively.
In vivo
studies, using i.p. administration, indicated that the parent, EpoB, was highly toxic to mice and showed little therapeutic effect when compared with a lead compound, dEpoB. More significantly, dEpoB (25–40 mg/kg, Q2Dx5, i.p.) showed far superior therapeutic effects and lower toxicity than paclitaxel, doxorubicin, camptothecin, or vinblastine (at maximal tolerated doses) in parallel experiments. For mammary adenocarcinoma xenografts resistant to adriamycin, MCF-7/Adr, superior therapeutic effects were obtained with dEpoB compared with paclitaxel when i.p. regimens were used. For ovarian adenocarcinoma xenografts, SK-OV-3, dEpoB (i.p.), and paclitaxel (i.v.) gave similar therapeutic effects. In nude mice bearing a human mammary carcinoma xenograft (MX-1), marked tumor regression and cures were obtained with dEpoB.
The adenovirus E3/10.4K–14.5K proteins down-modulate the apoptosis receptor Fas/Apo-1 by inducing its internalization
Authors: Elsing Andreas , Burgert Hans-Gerhard
Published: July 2002

Adenoviruses (Ads) have evolved multiple mechanisms to evade the host immune response. Several of the immunomodulatory Ad proteins are encoded in early transcription unit 3 (E3). The E3/19K protein interferes with antigen presentation and T cell recognition, whereas the E3/10.4K, 14.5K, and 14.7K proteins can protect cells from tumor necrosis factor α-mediated lysis. Here, we describe an additional activity of E3 proteins. Transfectants expressing all E3 proteins of Ad2 exhibit a profound reduction of the apoptosis receptor CD95 (Fas, APO-1) on the cell surface. In contrast, cells expressing only the E3A region have normal Fas levels. Thus, one of the E3B proteins (10.4K, 14.5K, or 14.7K) seems to be responsible for this effect. To identify the E3B products involved, each individual E3B ORF was selectively disrupted. Examination of stable cell lines containing the mutated E3 regions showed that Fas expression is restored when either the 10.4K or the 14.5K ORF is disrupted, whereas mutation of the 14.7K ORF does not rescue Fas expression. Loss of Fas on the cell surface is accompanied by a similar decrease of total Fas levels. However, in the presence of lysosomotropic agents Fas accumulates in endosomal/lysosomal vesicles, indicating that 10.4K–14.5K induce internalization and degradation of Fas. Down-regulation of Fas but not CD40 is also observed during infection and as a consequence, Ad-infected cells are protected from Fas-mediated apoptosis. Thus, the Fas system is implicated in Ad pathogenesis.
Mahogany
(
mg
) stimulates feeding and increases basal metabolic rate independent of its suppression of
agouti
Authors: Dinulescu Daniela M. , Fan Wei , Boston Bruce A. , McCall Kathleen , Lamoreux M. Lynn , Moore Karen J. , Montagno Jill , Cone Roger D.
Published: July 2002

The
mahogany
(
mg
) locus originally was identified as a recessive suppressor of
agouti
, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of
agouti
cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that
mg
, when homozygous, blocks not only the ability of
agouti
to induce a yellow coat color when expressed in the skin of the
lethal yellow
mouse (

A
Y

), but also the obesity resulting from ectopic expression of
agouti
in the brain. Detailed analysis of

mg/mg A
Y
/a

animals, presented here, demonstrates that
mg/mg
blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however,
mg/mg
did not reduce hyperphagia in the

A
Y
/a

mouse. Furthermore,
mg/mg
induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of

A
Y

. Consequently, although
mahogany
is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of
agouti
.
Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination
Authors: Giblin Michael F. , Wang Nannan , Hoffman Timothy J. , Jurisson Silvia S. , Quinn Thomas P.
Published: July 2002

α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys
4,10
,
d
-Phe
7
–α-MSH
4-13
analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding
99m
Tc- and
188
ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine.
In vivo
, the
99m
TcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via
99m
Tc and
188
Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.
A theory for controlling cell cycle dynamics using a reversibly binding inhibitor
Authors: Gardner Timothy S. , Dolnik Milos , Collins James J.
Published: July 2002

We demonstrate, by using mathematical modeling of cell division cycle (CDC) dynamics, a potential mechanism for precisely controlling the frequency of cell division and regulating the size of a dividing cell. Control of the cell cycle is achieved by artificially expressing a protein that reversibly binds and inactivates any one of the CDC proteins. In the simplest case, such as the checkpoint-free situation encountered in early amphibian embryos, the frequency of CDC oscillations can be increased or decreased by regulating the rate of synthesis, the binding rate, or the equilibrium constant of the binding protein. In a more complex model of cell division, where size-control checkpoints are included, we show that the same reversible binding reaction can alter the mean cell mass in a continuously dividing cell. Because this control scheme is general and requires only the expression of a single protein, it provides a practical means for tuning the characteristics of the cell cycle
in vivo
.
Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): Development of CRFR2β-selective antisauvagine-30
Authors: Rühmann Andreas , Bonk Ines , Lin Chijen R. , Rosenfeld Michael G. , Spiess Joachim
Published: July 2002

Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [
125
I-Tyr
0
]oCRF or [
125
I-Tyr
0
]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2β (mCRFR2β). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (
K
d
= 5.7 ± 1.6 nM) and mCRFR2β (
K
d
= 4.0 ± 2.3 nM), [
d
Phe
11
,His
12
]Svg
(11–40)
, [
d
Leu
11
]Svg
(11–40)
, [
d
Phe
11
]Svg
(11–40)
, and Svg
(11–40)
bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β. In agreement with the results of these binding experiments, [
d
Phe
11
,His
12
]Svg
(11–40)
, named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2β.
The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development
Authors: Kallunki Pekka , Edelman Gerald M. , Jones Frederick S.
Published: July 2002

The cell adhesion molecule L1 mediates axonal guidance during neural development and mutations in its gene result in severe neurological defects. In previous studies, we identified the promoter for the L1 gene and showed that a neural restrictive silencer element (NRSE) was critical for preventing ectopic expression of L1 during early embryonic development. In the present study, we have investigated the role of the NRSE in the regulation of L1 expression during postnatal development. In gel mobility shift experiments, the NRSE formed DNA–protein complexes with nuclear extracts prepared from the brains of postnatal mice. To examine the influence of the NRSE on postnatal patterns of L1 expression
in vivo
, we compared the expression of two
lacZ
transgene constructs, one containing the native L1 gene regulatory sequences (L1lacZ) and another (L1lacZΔN) lacking the NRSE. Newborn mice carrying the L1lacZΔN showed enhanced β-galactosidase expression relative to L1lacZ in the brain and ectopic expression in nonneural tissues. In contrast to L1lacZ mice, however, L1lacZΔN mice showed an unexpected loss, during postnatal development and in the adult, of β-galactosidase expression in several neural structures, including the neural retina, cerebellum, cortex, striatum, and hippocampus. These data support the conclusion that the NRSE not only plays a role in the silencing of L1 expression in nonneural tissues during early development but also can function as a silencer and an enhancer of L1 expression in the nervous system of postnatal and adult animals.
Dynamics of different functional parts of bacteriorhodopsin: H-
2
H labeling and neutron scattering
Authors: Réat Valérie , Patzelt Heiko , Ferrand Michel , Pfister Claude , Oesterhelt Dieter , Zaccai Giuseppe
Published: July 2002

We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen–deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in
Halobacterium salinarum
. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
Cellular memory: Neutrophil orientation reverses during temporally decreasing chemoattractant concentrations
Authors: Albrecht Eric , Petty Howard R.
Published: July 2002

Cell directional orientation or shape polarization is the first cellular step in neutrophil locomotion. To better understand how chemoattractants interact with cells, we studied neutrophil polarization (or shape changes) during exposure to a temporally decreasing chemoattractant signal ofN-formyl-methionyl-leucyl-phenylalanine (FMLP) in the absence of a spatial concentration gradient. To accomplish this objective, we used a manifold of differing FMLP concentrations attached to a stopped-flow microscope chamber. Spatial gradients of a fluorescent chemotactic peptide could not be detected in the chamber by using microfluorometry. When FMLP was injected at continually increasing concentrations at 10-s intervals, the shape and relative direction of the neutrophil persisted. However, when temporally decreasing FMLP concentrations were injected, ≈80% of the cells changed their direction with 44% of the total cells swinging about to 180° ± 15°. Most of these directional changes involved dissolution of both the lamellipodium and uropod and reformation of these structures 180° from their original positions. This research suggests that neutrophils reverse their morphological polarity when exposed to temporally decreasing ligand concentrations by “remembering” their ligand exposure history and relative direction.
The natural killer gene complex genetic locusChokencodes Ly-49D, a target recognition receptor that activates natural killing
Authors: Idris Azza H. , Smith Hamish R. C. , Mason Llewellyn H. , Ortaldo John R. , Scalzo Anthony A. , Yokoyama Wayne M.
Published: July 2002

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization ofChokin the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6–Cmv1rcongenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4+B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is theChokgene product and is a mouse NK cell receptor capable of directly triggering natural killing.
Low- and high-level transgenic expression of β
2
-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice
Authors: Dorn Gerald W. , Tepe Nicole M. , Lorenz John N. , Koch Walter J. , Liggett Stephen B.
Published: July 2002

Transgenic overexpression of Gαq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed β-adrenergic receptor (βAR) function. The role of βAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the βAR kinase (βARK), which acts to inhibit the kinase, or concomitant overexpression of the β
2
AR at low (≈30-fold, Gαq/β
2
AR
L
), moderate (≈140-fold, Gαq/β
2
AR
M
), and high (≈1,000-fold, Gαq/β
2
AR
H
) levels above background βAR density. Expression of the βARK inhibitor had no effect on the phenotype, consistent with the lack of increased βARK levels in Gαq mice. In marked contrast, Gαq/β
2
AR
L
mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and α-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized β
2
AR/AC signal cannot be excluded. Higher expression of receptors in Gαq/β
2
AR
M
mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With ≈1,000-fold overexpression, the majority of Gαq/β
2
AR
H
mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of βAR signaling is deleterious in heart failure, selective enhancement by overexpressing the β
2
AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.
The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule
Authors: Bose Himangshu S. , Whittal Randy M. , Baldwin Michael A. , Miller Walter L.
Published: July 2002

The steroidogenic acute regulatory protein (StAR) increases the movement of cholesterol from the outer to the inner membrane of adrenal and gonadal mitochondria, thus providing the substrate for steroid hormone biosynthesis. Deletion of 62 amino-terminal aa produces a cytoplasmic form of StAR (N-62 StAR) that lacks the mitochondrial leader sequence but retains full activity and appears to act at the outer mitochondrial membrane. At neutral pH the native state of bacterially expressed N-62 StAR protein displays cooperative unfolding under the influence of urea with ΔG
H2O
= −4.1 kcal/mol, and it remains correctly folded down to pH 4. Limited proteolysis at different pHs shows that the biologically essential C-terminal region is accessible to solvent, and that the N-terminal domain is compact at pH 8 and partially unfolds below pH 4. Secondary structural analysis of CD curves suggests that the unfolding may coincide with an increase in α-helical character at pH 3.5. Fluorescence spectroscopy at pH 3–8 and at 0–6 M urea is consistent with two distinct domains, a compact N-terminal domain containing tryptophans 96 and 147 and a more solvent-accessible C-terminal domain containing tryptophans 241 and 250. These observations suggest that StAR forms a molten globule structure at pH 3.5–4.0. As the mitochondrial proton pump results in an electrochemical gradient, and as StAR must unfold during mitochondrial entry, StAR probably undergoes a similar conformational shift to an extended structure while interacting with the mitochondrial outer membrane, allowing this apparent molten globule form to act as an on/off switch for cholesterol entry into the mitochondria.
Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA
Authors: O’Connor Michael , Asai Tsuneaki , Squires Catherine L. , Dahlberg Albert E.
Published: July 2002

The downstream box (DB) is a sequence element that enhances translation of several bacterial and phage mRNAs. It has been proposed that the DB enhances translation by base pairing transiently to bases 1469–1483 of 16S rRNA, the so-called anti-DB, during the initiation phase of translation. We have tested this model of enhancer action by constructing mutations in the anti-DB that alter its mRNA base-pairing potential and examining expression of a variety of DB-containing mRNAs in strains expressing the mutant anti-DB 16S rRNA. We found that the rRNA mutant was viable and that expression of all tested DB-containing mRNAs was completely unaffected by radical alterations in the proposed anti-DB. These findings lead us to conclude that enhancement of translation by the DB does not involve mRNA–rRNA base pairing.
Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient
ob/ob
mice
Authors: Seufert Jochen , Kieffer Timothy J. , Habener Joel F.
Published: July 2002

Leptin controls feeding behavior and insulin secretion from pancreatic β-cells. Insulin stimulates the production of leptin, thereby establishing an adipoinsular axis. Earlier we identified leptin receptors on pancreatic β-cells and showed leptin-mediated inhibition of insulin secretion by activation of ATP-sensitive potassium channels. Here we examine transcriptional effects of leptin on the promoter of the rat insulin I gene in rodent β-cells. A fall in levels of preproinsulin mRNA is detected
in vivo
in islets of
ob/ob
mice 24 h after a single injection of leptin, in isolated
ob/ob
islets treated with leptin
in vitro
and in the β-cell line INS-1 on leptin exposure when preproinsulin mRNA expression is stimulated by 25 mM glucose or 10 nM glucagon-like peptide 1. Under these conditions, transcriptional activity of −410 bp of the rat insulin I promoter is inhibited by leptin, whereas transactivation of a 5′-deleted promoter (−307 bp) is not. The −307 sequence contains the known glucose-responsive control elements (E2:A3/4). Constitutive activation of ATP-sensitive potassium channels by diazoxide does not alter leptin inhibition of preproinsulin mRNA levels. Distinct protein–DNA complexes appear on the rat insulin I promoter sequences located between −307 and −410 with nuclear extracts from
ob/ob
islets in response to leptin, including a signal transducer and activator of transcription (STAT)5b binding site. These results indicate that leptin inhibits transcription of the preproinsulin gene by altering transcription factor binding to sequences upstream from the elements (307 bp) that confer glucose responsivity to the rat insulin I gene promoter. Thus leptin exerts inhibitory effects on both insulin secretion and insulin gene expression in pancreatic β-cells, but by different cellular mechanisms.
Release of calcium from stores alters the morphology of dendritic spines in cultured hippocampal neurons
Authors: Korkotian E. , Segal M.
Published: July 2002

The ability to monitor ongoing changes in the shape of dendritic spines has important implications for the understanding of the functional correlates of the great variety of shapes and sizes of dendritic spines in central neurons. We have monitored and three-dimensionally reconstructed dendritic spines in cultured hippocampal neurons over several hours of observation in a confocal laser scanning microscope. In the absence of extrinsic stimulation, the dimensions of dendritic spines of 3-week-old cultured neurons did not change to any significant degree over 3–4 hr in the culture dish, unlike the case with younger cultures. Releasing calcium from stores with pulse application of caffeine causes a transient rise of [Ca
2+
]
i
in dendrites and spines, monitored with the calcium dye Oregon-green. Application of caffeine to a dendrite imaged with calcein caused a fast and significant increase in the size of existing dendritic spines and could lead to formation of new ones. This effect is mediated by calcium released from the ryanodine-sensitive stores, as application of caffeine in the presence of ryanodine blocked this effect on the morphology of dendritic spines. Thus, release of calcium from stores is sufficient to produce significant changes in the shape of dendritic spines of cultured hippocampal neurons.
RNA diversity has profound effects on the translation of neuronal nitric oxide synthase
Authors: Wang Yang , Newton Derek C. , Robb G. Brett , Kau Cheng-Lin , Miller Tricia L. , Cheung Anthony H. , Hall Anne V. , VanDamme Suzannah , Wilcox Josiah N. , Marsden Philip A.
Published: July 2002

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC
1.14.13.39
) mRNA species revealed
NOS1
to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5′-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5′-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5′-untranslated regions, which have profound effects on translation both
in vitro
and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.
Identification of lumichrome as a
Sinorhizobium
enhancer of alfalfa root respiration and shoot growth
Authors: Phillips Donald A. , Joseph Cecillia M. , Yang Guo-Ping , Martínez-Romero Esperanza , Sanborn James R. , Volpin Hanne
Published: July 2002

Sinorhizobium meliloti
bacteria produce a signal molecule that enhances root respiration in alfalfa (
Medicago sativa
L.) and also triggers a compensatory increase in whole-plant net carbon assimilation. Nuclear magnetic resonance, mass spectrometry, and ultraviolet–visible absorption identify the enhancer as lumichrome, a common breakdown product of riboflavin. Treating alfalfa roots with 3 nM lumichrome increased root respiration 21% (
P
< 0.05) within 48 h. A closely linked increase in net carbon assimilation by the shoot compensated for the enhanced root respiration. For example, applying 5 nM lumichrome to young alfalfa roots increased plant growth by 8% (
P
< 0.05) after 12 days. Soaking alfalfa seeds in 5 nM lumichrome before germination increased growth by 18% (
P
< 0.01) over the same period. In both cases, significant growth enhancement (
P
< 0.05) was evident only in the shoot.
S. meliloti
requires exogenous CO
2
for growth and may benefit directly from the enhanced root respiration that is triggered by lumichrome. Thus
Sinorhizobium
–alfalfa associations, which ultimately form symbiotic N
2
-reducing root nodules, may be favored at an early developmental stage by lumichrome, a previously unrecognized mutualistic signal. The rapid degradation of riboflavin to lumichrome under many physiological conditions and the prevalence of riboflavin release by rhizosphere bacteria suggest that events demonstrated here in the
S. meliloti
–alfalfa association may be widely important across many plant–microbe interactions.
Altered emotional and locomotor responses in mice deficient in the transcription factor CREM
Authors: Maldonado Rafael , Smadja Claire , Mazucchelli Cristina , Sassone-Corsi Paolo
Published: July 2002

Various transcription factors act as nuclear effectors of the cAMP-dependent signaling pathway. These are the products of three genes in the mouse, CREB, CRE modulator (CREM), and ATF-1. CREM proteins are thought to play important roles within the hypothalamic–pituitary axis and in the control of rhythmic functions in the pineal gland. We have generated CREM-mutant mice and investigated their response in a variety of behavioral tests. CREM-null mice show a drastic increase in locomotion. In contrast to normal mice, the CREM-deficient mice show equal locomotor activity during the circadian cycle. The anatomy of the hypothalamic suprachiasmatic nuclei, the center of the endogenous pacemaker, is normal in mutant mice. Remarkably, CREM mutant mice also elicit a different emotional state, revealed by a lower anxiety in two different behavioral models, but they preserve the conditioned reactiveness to stress. These results demonstrate the high degree of functional specificity of each cAMP-responsive transcription factor in behavioral control.
Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury
Authors: Ma Xin L. , Gao Feng , Liu Gao-Lin , Lopez Bernard L. , Christopher Theodore A. , Fukuto Jon M. , Wink David A. , Feelisch Martin
Published: July 2002

Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO
−
) and nitric oxide (NO
⋅
), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 μmol/kg
S-
nitrosoglutathione (GSNO) (an NO
⋅
donor), or 3 μmol/kg Angeli’s salt (AS) (a source of NO
−
) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO
−
to NO
⋅
, completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO
⋅
is protective, NO
−
increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.
A finding of Oligocene primates on the European continent
Authors: Köhler Meike , Moyà-Solà Salvador
Published: July 2002

In this paper, we provide evidence that contrary to the current view, primates on the European continent did survive the dramatic extinction/origination event across the Eocene/Oligocene boundary 34 million years ago that severely affected the Eurasian mammal communities (the European “Grande Coupure” and the Asian “Mongolian Remodeling”). The survival of a mouse-sized omomyid for at least 2 million years, recorded in two localities of the Lower Stampian (Lower Oligocene) in a well dated stratigraphic series of fluviatile sediments in the north oriental sector of the Ebro Basin (Northeastern Spain), reflects the size-related survival pattern described recently for other coeval mammalian taxa.