bluuebunny/crossref_metadata_2025 huggingface dataset:
A genetically clamped renin transgene for the induction of hypertension
Authors: Caron Kathleen M. I. , James Leighton R. , Kim Hyung-Suk , Morham Scott G. , Lopez Maria Luisa S. Sequeira , Gomez R. Ariel , Reudelhuber Timothy L. , Smithies Oliver
Published: July 2002

Experimental analysis of the effects of individual components of complex mammalian systems is frequently impeded by compensatory adjustments that animals make to achieve homeostasis. We here introduce a genetic procedure for eliminating this type of impediment, by using as an example the development and testing of a transgene for “genetically clamping” the expression of renin, the major homeostatically responding component of the renin–angiotensin system, one of the most important regulators of blood pressure. To obtain a renin transgene whose expression is genetically clamped at a constant level, we have used single-copy chosen-site gene targeting to insert into a liver-specific locus a single copy of a modified mouse renin transgene driven by a liver-specific promoter/enhancer. The resulting transgene expresses renin ectopically at a constant high level in the liver and leads to elevated plasma levels of prorenin and active renin. The transgenic mice display high blood pressure, enhanced thirst, high urine output, proteinuria, and kidney damage. Treatment with the angiotensin II type I receptor antagonist, losartan, reduces the hypertension, albuminuria, and kidney damage, but does not affect expression of the transgene. This genetically clamped renin transgene can be used in models in which hypertension and its complications need to be investigated in a high prorenin/renin environment that is not subject to homeostatic compensations by the animal when other factors are changed.
Structural basis for the interaction of the fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS) with the antibiotic target MurA
Authors: Schönbrunn Ernst , Eschenburg Susanne , Luger Karolin , Kabsch Wolfgang , Amrhein Nikolaus
Published: July 2002

The extrinsic fluorescence dye 8-anilino-1-naphthalene sulfonate (ANS) is widely used for probing conformational changes in proteins, yet no detailed structure of ANS bound to any protein has been reported so far. ANS has been successfully used to monitor the induced-fit mechanism of MurA [UDPGlcNAc
enol
pyruvyltransferase (EC
2.5.1.7
)], an essential enzyme for bacterial cell wall biosynthesis. We have solved the crystal structure of the ANS⋅MurA complex at 1.7-Å resolution. ANS binds at an originally solvent-exposed region near Pro-112 and induces a major restructuring of the loop Pro-112–Pro-121, such that a specific binding site emerges. The fluorescence probe is sandwiched between the strictly conserved residues Arg-91, Pro-112, and Gly-113. Substrate binding to MurA is accompanied by large movements especially of the loop and Arg-91, which explains why ANS is an excellent sensor of conformational changes during catalysis of this pharmaceutically important enzyme.
Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions
Authors: Han Tae H. , Hyduke Daniel R. , Vaughn Mark W. , Fukuto Jon M. , Liao James C.
Published: July 2002

Understanding the interaction of nitric oxide (NO) with red blood cells (RBCs) is vital to elucidating the metabolic fate of NO in the vasculature. Because hemoglobin (Hb) is the most abundant intraerythrocytic protein and reacts rapidly with NO, the interaction of NO with Hb has been studied extensively. We and others have shown the NO reaction with RBCs is nearly 1,000-fold slower than the reaction with cell-free Hb. Because the reaction rate of NO with cell-free Hb and RBCs is quite different, we hypothesize that different reaction products evolve under locally high NO concentrations, which can be generated by bolus NO addition or NO inhalation. Here we use electron paramagnetic resonance to show that bolus NO addition to cell-free Hb solutions results in nitrosylhemoglobin [HbFe(II)NO] formation as a minor product through a MetHb-dependent pathway. Further, the RBC is shown to be more prone to form HbFe(II)NO under this heterogeneous condition compared with an equivalent free-Hb solution. In both cases, trapping MetHb with cyanide blocked the formation of HbFe(II)NO. We conclude that the formation of HbFe(II)NO is a heterogeneous phenomenon involving three successive reactions of NO with the same heme molecule. These results were supported further by mathematically modeling NO–Hb reactions and diffusion.
High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment
Authors: Wu Anna M. , Yazaki Paul J. , Tsai Shih-wa , Nguyen Khoi , Anderson Anne-Line , McCarthy Deborah W. , Welch Michael J. , Shively John E. , Williams Lawrence E. , Raubitschek Andrew A. , Wong Jeffrey Y. C. , Toyokuni Tatsushi , Phelps Michael E. , Gambhir Sanjiv S.
Published: July 2002

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C
H
3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-
N,N′,N′′, N′′′
-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (
t
1/2
= 12.7 h).
In vivo
distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with
64
Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram ± 3.79) compared with control tumor (6.0% injected dose per gram ± 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3–4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.
Reproductive aging and mating: The ticking of the biological clock in female cockroaches
Authors: Moore Patricia J. , Moore Allen J.
Published: July 2002

Females are expected to have different mating preferences because of the variation in costs and benefits of mate choice both between females and within individual females over a lifetime. Workers have begun to look for, and find, the expected variation among females in expressed mating preferences. However, variation within females caused by changes in intrinsic influences has not been examined in detail. Here we show that reproductive aging caused by delayed mating resulted in reduced choosiness by female
Nauphoeta cinerea
, a cockroach that has reproductive cycles and gives live birth. Male willingness to mate was unaffected by variation in female age. Females who were beyond the optimal mating age, 6 days postadult molt, required considerably less courtship than their younger counterparts. Females who were older when they mated had fewer offspring per clutch and fewer clutches than females who mated young. Thus, reduced choosiness was correlated with a permanent reduction in fertility. There was no difference in overall senescence among females, and thus the reduction in clutch size did not result in the expected increased lifespan. We suggest that reproductive aging in
N. cinerea
, similar to aging in general, occurs because the maintenance of oocytes is costly, and selection is relaxed after the optimal mating period. Our results further suggest that selection for continued choosiness is also relaxed and supports direct selection on female choosiness and a cost to choosiness.
Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition
Authors: Bauer Stefan , Kirschning Carsten J. , Häcker Hans , Redecke Vanessa , Hausmann Susanne , Akira Shizuo , Wagner Hermann , Lipford Grayson B.
Published: July 2002

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably “gain of function” to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.
Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21
WAF1/CIP1
expression in keratinocyte differentiation
Authors: Santini Maria Paola , Talora Claudio , Seki Toshihiko , Bolgan Loretta , Dotto G. Paolo
Published: July 2002

Calcium functions as a trigger for the switch between epithelial
cell growth and differentiation. We report here that the
calcium/calmodulin-dependent phosphatase calcineurin is
involved in this process. Treatment of primary mouse keratinocytes with
cyclosporin A, an inhibitor of calcineurin activity, suppresses the
expression of terminal differentiation markers and of
p21
WAF1/Cip1
and p27
KIP1
, two cyclin-dependent
kinase inhibitors that are usually induced with differentiation. In
parallel with down-modulation of the endogenous genes,
suppression of calcineurin function blocks induction of the promoters
for the p21
WAF1/Cip1
and loricrin differentiation marker
genes, whereas activity of these promoters is enhanced by calcineurin
overexpression. The calcineurin- responsive region of the p21
promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA
box, and calcineurin induces activity of the p21 promoter through
Sp1/Sp3-dependent transcription. We find that the
endogenous NFAT-1 and -2 transcription factors, major
downstream targets of calcineurin, associate with Sp1 in keratinocytes
in a calcineurin-dependent manner, and calcineurin up-regulates
Sp1/Sp3-dependent transcription and p21 promoter activity in
synergism with NFAT1/2. Thus, our study reveals an important role for
calcineurin in control of keratinocyte differentiation and p21
expression, and points to a so-far-unsuspected interconnection among
this phosphatase, NFATs, and Sp1/Sp3-dependent transcription.
A neurohistochemical blueprint for pain-induced loss of appetite
Authors: Malick Amy , Jakubowski Moshe , Elmquist Joel K. , Saper Clifford B. , Burstein Rami
Published: July 2002

A common complaint among pain patients is that they lose their
appetite. These accounts are anecdotal, however, and the neural
mechanism underlying pain-induced loss of appetite remains unknown. In
this study, we documented the occurrence of appetite loss in patients
under migraine attack and investigated the neuronal substrate of
pain-induced anorexia in our animal model of intracranial pain. We
found that loss of appetite during the migraine attack in humans
coincided strongly with the onset and duration of the head pain in
32/39 cases, and that brief noxious stimulation of the dura in
conscious rats produced a transient suppression of food intake. Mapping
of neuronal activation in the rat showed that noxious dural stimulation
induced a 3- to 4-fold increase in the number of Fos-positive neurons
in medullary dorsal horn areas that process nociceptive signals
(laminae I, V) and in parabrachial and hypothalamic neurons positioned
to suppress feeding behavior. In the parabrachial area, activated
neurons were localized in the superior-lateral subnucleus, and 40% of
them expressed the mRNA encoding the anorectic neuropeptide
cholecystokinin. In the hypothalamus, activated Fos-positive neurons
were found in the dorsomedial area of the ventromedial nucleus, and
76% of them expressed the mRNA for cholecystokinin type-B receptor.
Based on these findings, we suggest that at least one of several groups
of hypothalamic neurons that normally inhibit appetite in response to
metabolic cues is positioned to mediate the suppression of food intake
by pain signals.
Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis
Authors: Heinze Katrin G. , Koltermann Andre , Schwille Petra
Published: July 2002

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease
Eco
RI.
Arousal effect of orexin A depends on activation of the histaminergic system
Authors: Huang Zhi-Li , Qu Wei-Min , Li Wei-Dong , Mochizuki Takatoshi , Eguchi Naomi , Watanabe Takeshi , Urade Yoshihiro , Hayaishi Osamu
Published: July 2002

Orexin neurons are exclusively localized in the lateral
hypothalamic area and project their fibers to the entire central
nervous system, including the histaminergic tuberomammillary nucleus
(TMN). Dysfunction of the orexin system results in the sleep disorder
narcolepsy, but the role of orexin in physiological sleep–wake
regulation and the mechanisms involved remain to be elucidated. Here we
provide several lines of evidence that orexin A induces wakefulness by
means of the TMN and histamine H
1
receptor (H1R). Perfusion
of orexin A (5 and 25 pmol/min) for 1 hr into the TMN of rats
through a microdialysis probe promptly increased wakefulness for 2
hr after starting the perfusion by 2.5- and 4-fold, respectively,
concomitant with a reduction in rapid eye movement (REM) and non-REM
sleep. Microdialysis studies showed that application of orexin A to the
TMN increased histamine release from both the medial preoptic area and
the frontal cortex by ≈2-fold over the baseline for 80 to 160 min in
a dose-dependent manner. Furthermore, infusion of orexin A (1.5
pmol/min) for 6 hr into the lateral ventricle of mice produced a
significant increase in wakefulness during the 8 hr after starting
infusion to the same level as the wakefulness observed during the
active period in wild-type mice, but not at all in H1R gene knockout
mice. These findings strongly indicate that the arousal effect of
orexin A depends on the activation of histaminergic neurotransmission
mediated by H1R.
Electrostatics of nanosystems: Application to microtubules and the ribosome
Authors: Baker Nathan A. , Sept David , Joseph Simpson , Holst Michael J. , McCammon J. Andrew
Published: July 2002

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.
Intensely pleasurable responses to music correlate with activity in brain regions implicated in reward and emotion
Authors: Blood Anne J. , Zatorre Robert J.
Published: July 2002

We used positron emission tomography to study neural mechanisms
underlying intensely pleasant emotional responses to music.
Cerebral blood flow changes were measured in response to
subject-selected music that elicited the highly pleasurable
experience of “shivers-down-the-spine” or “chills.”
Subjective reports of chills were accompanied by changes in heart rate,
electromyogram, and respiration. As intensity of these chills
increased, cerebral blood flow increases and decreases were observed in
brain regions thought to be involved in reward/motivation, emotion,
and arousal, including ventral striatum, midbrain, amygdala,
orbitofrontal cortex, and ventral medial prefrontal cortex. These brain
structures are known to be active in response to other
euphoria-inducing stimuli, such as food, sex, and drugs of abuse. This
finding links music with biologically relevant, survival-related
stimuli via their common recruitment of brain circuitry involved in
pleasure and reward.
Modulation of specific intestinal epithelial progenitors by enteric neurons
Authors: Bjerknes Matthew , Cheng Hazel
Published: July 2002

The proglucagon-derived peptide glucagon-like peptide 2 (GLP-2), a product of a subset of gut epithelial cells, is pursued clinically for its ability to stimulate gut epithelial growth and repair. Here we show that although specific epithelial progenitors respond to GLP-2 administration, the epithelium does not express the GLP-2 receptor. Rather, enteric neurons express the receptor, respond to GLP-2, and transmit a signal (which can be blocked by the voltage-gated sodium channel inhibitor tetrodotoxin) back to the epithelium. Thus the nervous system is a key component of a feedback loop regulating epithelial growth and repair.
Intermediates in V(D)J recombination: A stable RAG1/2 complex sequesters cleaved RSS ends
Authors: Jones Jessica M. , Gellert Martin
Published: July 2002

Rearrangement of gene segments to generate antigen receptor coding regions depends on the RAG1/2 recombinase, which assembles a synaptic complex between two DNA signal sequences and then cleaves the DNA directly adjacent to the paired signals. After coupled cleavage of complementary signal sequences, virtually all of the cleaved signal ends remained associated with RAG1/2 in stable complexes. These signal end complexes were distinct from various precleavage RAG1/2 signal complexes in that they were resistant to treatment with heparin. A mammalian joining apparatus consisting of purified Ku70/86, XRCC4, and DNA ligase IV proteins was sufficient to join deproteinized cleaved ends, but retention of signal sequences within the signal end complex blocked access to the DNA ends and prevented their joining by these proteins. Sequestration of cleaved ends within the signal end complex would account for the persistence of these ends in the cell after cleavage and may explain why they do not normally activate the DNA-damage-dependent cell cycle checkpoint.
Coevolution of head, neck, and tail domains of myosin heavy chains
Authors: Korn Edward D.
Published: July 2002

Myosins, a large family of actin-based motors, have one or two heavy chains with one or more light chains associated with each heavy chain. The heavy chains have a (generally) N-terminal head domain with an ATPase and actin-binding site, followed by a neck domain to which the light chains bind, and a C-terminal tail domain through which the heavy chains self-associate and/or bind the myosin to its cargo. Approximately 140 members of the myosin superfamily have been grouped into 17 classes based on the sequences of their head domains. I now show that a phylogenetic tree based on the sequences of the combined neck and tail domains groups 144 myosins, with a few exceptions, into the same 17 classes. For the nine myosin classes that have multiple members, phylogenetic trees based on the head domain or the combined neck/tail domains are either identical or very similar. For class II myosins, very similar phylogenetic trees are obtained for the head, neck, and tail domains of 47 heavy chains and for 29 essential light chains and 19 regulatory light chains. These data strongly suggest that the head, neck, and tail domains of all myosin heavy chains, and light chains at least of class II myosins, have coevolved and are likely to be functionally interdependent, consistent with biochemical evidence showing that regulated actin-dependent MgATPase activity ofDictyosteliummyosin II requires isoform specific interactions between the heavy chain head and tail and light chains.
PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine
Authors: Okazaki Taku , Maeda Akito , Nishimura Hiroyuki , Kurosaki Tomohiro , Honjo Tasuku
Published: July 2002

PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (FcγRIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca
2+
mobilization, and tyrosine phosphorylation of effector molecules, including Igβ, Syk, phospholipase C-γ2 (PLCγ2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling.
The neural representation of postural control in humans
Authors: Karnath Hans-Otto , Ferber Susanne , Dichgans Johannes
Published: July 2002

Lesion of the “vestibular cortex” in the human posterior
insula leads to a tilted perception of visual vertical but not to
tilted body posture and loss of lateral balance. However, some stroke
patients show the reverse pattern. Although their processing of visual
and vestibular inputs for orientation perception of the visual world is
undisturbed, they push away actively from the ipsilesional side (the
side of lesion location), leading to a contraversive tilt of the body
(tilt toward the side opposite to the lesion) and falling to that side.
Recently, the origin of contraversive pushing was identified as an
altered perception of the body's orientation in relation to gravity.
These patients experience their body as oriented “upright” when
actually tilted enormously to the ipsilesional side (18° on average).
The findings argued for a separate pathway in humans for sensing body
orientation in relation to gravity apart from the one projecting to the
vestibular cortex. The present study aimed at identifying this brain
area. The infarcted brain regions of 23 consecutively admitted patients
with severe contraversive pushing were projected onto a template MRI
scan, which had been normalized to Talairach space. The overlapping
area of these infarctions centered on the posterolateral thalamus. Our
finding necessitates reinterpretation of this area as being only a
“relay structure” of the vestibular pathway on its way from the
brainstem to the vestibular cortex. The ventral posterior and lateral
posterior nuclei of the posterolateral thalamus (and probably its
cortical projections) rather seem to be fundamentally involved in the
neural representation of a second graviceptive system in humans
decisive for our control of upright body posture.
SST3-selective potent peptidic somatostatin receptor antagonists
Authors: Reubi Jean Claude , Schaer Jean-Claude , Wenger Sandra , Hoeger Carl , Erchegyi Judit , Waser Beatrice , Rivier Jean
Published: July 2002

A family of octapeptide derivatives of somatostatin cyclized via a disulfide bridge (des-AA1,2,4,5,12,13[d-2Nal8]-somatostatin-14, ODN-8) was identified that has high affinity and selectivity for the human sst3somatostatin receptor subtype transfected in CCL39 cells. The binding affinity of carbamoyl-des-AA1,2,4,5,12,13[d-Cys3,Tyr7,d-Agl8(Me,2-naphthoyl)]-somatostatin-14 (sst3-ODN-8) is equal to that of somatostatin-28 for sst3and less than one-thousandth that for the other four somatostatin receptor subtypes. Compound sst3-ODN-8 potently reverses the somatostatin-28-induced inhibition of forskolin-stimulated cAMP production (pKB= 9.07) and reverses the somatostatin-28-induced stimulation of phospholipase C activity (pKi= 9.22) in sst3-transfected CCL39 cells. [125I-Tyr7]sst3-ODN-8 selectively labels sst3-expressing cells with subnanomolar binding affinity (KD= 0.27 nM). With the use of this radioligand, sst3-expressing human tumors, particularly inactive pituitary adenomas, can be identified with receptor autoradiography; moreover, areas of the human lymphoreticular system express sst3binding sites selectively displaced by nanomolar concentrations of sst3-ODN-8. Based on the structure–activity relationship of selected analogs substituted at positions 3, 7, and 8, we hypothesize that the basis for sst3selectivity, high affinity, and possibly antagonism resides in the ring size of the analog and the unique conformational and structural character of the N-methylated amino-2-naphthoyl side chain of aminoglycine at position 8 and not in the Tyr7substitution or in thed-configuration at position 3. The family of labeled and unlabeled sst3-ODN-8 analogs represents highly innovative, potent, and specific sst3-selective antagonist tools for the study of sst3-mediated physiological and pathophysiological conditions that may suggest novel clinical applications.
T-bet is rapidly induced by interferon-γ in lymphoid and myeloid cells
Authors: Lighvani Andre A. , Frucht David M. , Jankovic Dragana , Yamane Hidehiro , Aliberti Julio , Hissong Bruce D. , Nguyen Bai V. , Gadina Massimo , Sher Alan , Paul William E. , O'Shea John J.
Published: July 2002

Differentiation of naive CD4
+
T cells into
IFN-γ-producing T helper 1 (T
H
1) cells is pivotal
for protective immune responses against intracellular pathogens. T-bet,
a recently discovered member of the T-box transcription factor family,
has been reported to play a critical role in this process, promoting
IFN-γ production. Although terminal T
H
1 differentiation
occurs over days, we now show that challenge of mice with a
prototypical T
H
1-inducing stimulus,
Toxoplasma
gondii
soluble extract, rapidly induced IFN-γ and T-bet;
T-bet induction was substantially lower in IFN-γ-deficient mice.
Naive T cells expressed little T-bet, but this transcription factor was
induced markedly by the combination of IFN-γ and cognate antigen.
Human myeloid antigen-presenting cells showed T-bet induction after
IFN-γ stimulation alone, and this induction was antagonized by IL-4
and granulocyte/macrophage colony-stimulating factor. Although
T-bet was induced rapidly and directly by IFN-γ, it was not induced
by IFN-α, lipopolysaccharide, or IL-1, indicating that this action of
IFN-γ was specific. Moreover, T-bet induction was dependent on Stat1
but not Stat4. These data argue for a model in which IFN-γ gene
regulation involves an autocrine loop, whereby the cytokine regulates a
transcription factor that promotes its own production. These findings
substantially alter the current view of T-bet in IFN-γ regulation and
promotion of cell-mediated immune responses.
Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.
Authors: Bard M , Bruner D A , Pierson C A , Lees N D , Biermann B , Frye L , Koegel C , Barbuch R
Published: July 2002

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.
Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.
Authors: Vaz A D , Pernecky S J , Raner G M , Coon M J
Published: July 2002

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.
Expression of galectin-3 modulates T-cell growth and apoptosis.
Authors: Yang R Y , Hsu D K , Liu F T
Published: July 2002

Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.
Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.
Authors: Lauter F R , Ninnemann O , Bucher M , Riesmeier J W , Frommer W B
Published: July 2002

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.
Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.
Authors: Bastiaens P I , Jovin T M
Published: July 2002

We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.
Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.
Authors: Stilgenbauer S , Liebisch P , James M R , Schröder M , Schlegelberger B , Fischer K , Bentz M , Lichter P , Döhner H
Published: July 2002

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.
Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.
Authors: Hopwood B , Dalton S
Published: July 2002

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.
Functional organization of the hippocampal memory system
Authors: Eichenbaum Howard , Schoenbaum Geoffrey , Young Brian , Bunsey Michael
Published: July 2002

In humans declarative or explicit memory is supported by the
hippocampus and related structures of the medial temporal lobe working
in concert with the cerebral cortex. This paper reviews our progress in
developing an animal model for studies of cortical–hippocampal
interactions in memory processing. Our findings support the view that
the cortex maintains various forms of memory representation and that
hippocampal structures extend the persistence and mediate the
organization of these codings. Specifically, the parahippocampal
region, through direct and reciprocal interconnections with the cortex,
is sufficient to support the convergence and extended persistence of
cortical codings. The hippocampus itself is critical to the
organization cortical representations in terms of relationships among
items in memory and in the flexible memory expression that is the
hallmark of declarative memory.
Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.
Authors: Fehlbaum P , Bulet P , Chernysh S , Briand J P , Roussel J P , Letellier L , Hetru C , Hoffmann J A
Published: July 2002

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.
Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B.
Authors: Quan L T , Tewari M , O'Rourke K , Dixit V , Snipas S J , Poirier G G , Ray C , Pickup D J , Salvesen G S
Published: July 2002

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.
Temporal events in cyclopean vision.
Authors: Andrews T J , White L E , Binder D , Purves D
Published: July 2002

The majority of neurons in the primary visual cortex of primates can be activated by stimulation of either eye; moreover, the monocular receptive fields of such neurons are located in about the same region of visual space. These well-known facts imply that binocular convergence in visual cortex can explain our cyclopean view of the world. To test the adequacy of this assumption, we examined how human subjects integrate binocular events in time. Light flashes presented synchronously to both eyes were compared to flashes presented alternately (asynchronously) to one eye and then the other. Subjects perceived very-low-frequency (2 Hz) asynchronous trains as equivalent to synchronous trains flashed at twice the frequency (the prediction based on binocular convergence). However, at higher frequencies of presentation (4-32 Hz), subjects perceived asynchronous and synchronous trains to be increasingly similar. Indeed, at the flicker-fusion frequency (approximately 50 Hz), the apparent difference between the two conditions was only 2%. We suggest that the explanation of these anomalous findings is that we parse visual input into sequential episodes.
CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.
Authors: Cohen P S , Nakshatri H , Dennis J , Caragine T , Bianchi M , Cerami A , Tracey K J
Published: July 2002

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.
Discovery of the smallest Fayum Egyptian primates (Anchomomyini, Adapidae)
Authors: Simons Elwyn L.
Published: July 2002

Two new adapiform primate species from locality 41, Jebel Qatrani Formation, Egypt, are described. The first, here named
Wadilemur elegans
genus novum species novum (holotype Cairo Geological Museum 42211), consists of a right mandible with P
3
–M
3
. The second is assigned to
Anchomomys milleri
species novum, with a holotype Cairo Geological Museum 42842, that includes the right mandible with lower canine to M
3
. Both species are allied closely with genera that are known to be from Eocene deposits either in Europe, Tunisia, or the Sultanate of Oman (Arabia), thus enhancing earlier paleomagnetic evidence that locality 41 was deposited in Eocene times.