bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium/calmodulin‐dependent kinase II is involved in the facilitating effect of clozapine on NMDA‐ and electrically evoked responses in the medial prefrontal cortical pyramidal cells
Authors: Ninan Ipe , Jardemark Kent E. , Liang Xiaofu , Wang Rex Y.
Published: January 2003

Using the method of intracellular recording in in vitro brain slices, we investigated whether calcium/calmodulin‐dependent kinase II (CaMKII) is involved in the facilitating action produced by the atypical antipsychotic drug (APD) clozapine on N‐methyl‐D‐aspartate (NMDA)‐induced inward currents and electrically evoked excitatory postsynaptic currents (EPSCs) in pyramidal cells of the medial prefrontal cortex (mPFC). The CaMKII inhibitor, KN‐93 (N‐[2‐(N‐(4‐Chlorocinnamyl)‐N‐methylaminomethyl)phenyl]‐N‐[2‐hydroxyethyl]‐4‐methoxybenzenesulfonamide), but not the inactive isomer, KN‐92 (2‐[N‐(4‐Methoxybenzenesulfonyl)]amino‐N‐(4‐chlorocinnamyl)‐N‐methylbenzylamine, phosphate), blocked clozapine's augmenting effect on NMDA‐evoked responses in pyramidal cells of the rat mPFC. KN‐93 also inhibited the facilitatory effect of clozapine on electrically evoked responses in the pyramidal cells, while KN‐92 did not show any effect. Similarly, the calmodulin antagonist W‐7 (N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide) inhibited the augmenting effect of clozapine on NMDA‐ and electrically evoked responses in the pyramidal cells. To further test the role of CaMKII in mediating the augmenting action of clozapine, we performed experiments in alpha‐CaMKII mutant and wild‐type mice. In contrast to results in pyramidal cells from rats or wild‐type mice, clozapine was not able to potentiate NMDA‐induced currents in the mPFC pyramidal cells from the CaMKII mutant mouse. Both KN‐93 and W‐7, but not KN‐92, inhibited the augmenting action of clozapine in the pyramidal cells of wild‐type mice. Taken together, these results suggest that the facilitating action of clozapine on the NMDA‐ and electrically evoked responses in pyramidal cells of the mPFC requires activation of CaMKII enzyme. Synapse 47:285–294, 2003. © 2003 Wiley‐Liss, Inc.
Identification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine‐containing model peptide
Authors: Reichardt Peter , Schreiber André , Wichmann Gunnar , Metzner Gerhard , Efer Juergen , Raabe Ferdinand
Published: January 2003

Formation of in vitro adducts between different classes of xenobiotics and the lysine‐containing peptide Lys‐Tyr was monitored by high‐performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene‐2,4‐diisocyanate, 2,4‐dinitro‐1‐fluorobenzene, 2,4,6‐trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 29–36, 2003.
An empirical taxonomy for quality management systems: a study of the Hong Kong electronics industry
Authors: Yeung A.C.L , Chan L.Y , Lee T.S
Published: January 2003

This study investigates the existence of different patterns of quality management systems (QMS) and the relationship between such patterns and organizational performance by conducting a quantitative and qualitative study of 225 international and local firms in the electronics industry in Hong Kong. A cluster analysis of the survey data results in the identification of four patterns of QMS, which are labeled as undeveloped, frame, accommodating and strategic, respectively, according to the characteristics that each pattern displays. These four types of QMS were found to be associated with various organizational performance measures according to the stage of their development. The study suggests that organizations can improve their time‐based operational performance by establishing a frame quality system. However, the overall performance of an electronics company can be enhanced only by the establishment of a strategic quality system (SQS), which requires the involvement of top management. Our investigation further suggests that the development of a QMS is influenced by top management’s view on quality management. If quality management is viewed as an assurance system or a defensive strategy for answering customer requirements, a more advanced system will not be developed. The taxonomy developed here also enables researchers to understand the differences of major functions of quality systems at various stages of their development, the obstacles that management faces in the transformation process, and the corresponding strategies to be adopted.
The impact of human resource management practices on operational performance: recognizing country and industry differences
Authors: Ahmad Sohel , Schroeder Roger G.
Published: January 2003

The interest in strategic human resource management (HRM) has spawned a number of empirical research studies that investigated the impact of HRM practices on organizational performance. However, very little attention has been paid to address the impact of HRM practices on operations management and to generalize the findings across countries and industries. Success of some business decisions (e.g. globalization and merger and acquisition) necessitates recognition and reconciliation of the differences among HRM practices in different countries and industries. This study attempts to generalize the efficacy of seven HRM practices proposed by Pfeffer in the context of country and industry, focusing primarily on the effects of these practices on operations. The findings provide overall support for Pfeffer’s seven HRM practices and empirically validate an ideal‐type HRM system for manufacturing plants.
UNIQUENESS OF BUBBLE-FREE SOLUTION IN LINEAR RATIONAL EXPECTATIONS MODELS
Authors: Desgranges Gabriel , Gauthier Stéphane
Published: January 2003

One usually identifies bubble solutions to linear rational expectations models by extra components (irrelevant lags) arising in addition to market fundamentals. Although there are still many solutions relying on a minimal set of state variables, i.e., relating in equilibrium the current state of the economic system to as many lags as initial conditions, there is a conventional wisdom that the bubble-free (fundamentals) solution should be unique. This paper examines the existence of endogenous stochastic sunspot fluctuations close to solutions relying on a minimal set of state variables, which provides a natural test for identifying bubble and bubble-free solutions. It turns out that only one solution is locally immune to sunspots, independently of the stability properties of the perfect-foresight dynamics. In the standard saddle-point configuration for these dynamics, this solution corresponds to the so-called saddle stable path.
THE REAL-INTEREST-RATE GAP AS AN INFLATION INDICATOR
Authors: Neiss Katharine S. , Nelson Edward
Published: January 2003

A long-standing area of research and policy interest is the construction of a measure of monetary policy stance. One measure that has been proposed, as an alternative to indices that employ monetary aggregates or exchange rates, is the spread between the actual real interest rate and its flexible-price, or natural-rate, counterpart. We examine the properties of the natural real interest rate and real-interest-rate gap using a dynamic stochastic general equilibrium model. Issues we investigate include the response of the gap and its components to fundamental economic shocks and the indicator and forecasting properties of the real-interest-rate gap for inflation, both in the model and in the data. Our results suggest that the real-interest-rate gap has value as an inflation indicator, supporting a neo-Wicksellian framework.
An Incommensurate Superstructure of Hexagonal Tungsten Bronze
Authors: Bando Yoshio , Iijima Sumio
Published: January 2003

Incommensurate superstructures are interesting problems for high
resolution electron microscopy. If modulations are formed within a plane of
a host lattice parallel to the incident beam direction, their structures can
be known directly from images (1,2,3). In this paper, an incommensurate
superstructure of hexagonal potassium tungsten bronze,
K0.3WO3,
is observed by a 100B high-resolution electron microscope and its structure
model is proposed. The hexagonal tungsten bronze was determined by Magneli
(4) and its structure is shown in Fig. 1. The structure consists of
WO6 octahedra and alkali metal ions. The alkali
metal ions situated in hexagonal tunnels do not lie at the same level of the
WO6 octahedra but at ¼ c above or below them. It
was assumed that the alkali metal ions were distributed in a random fashion.
Fig. 2 shows a structure image of
K0.3WO3,
taken along the [100] direction. The white spots correspond to hexagonal
tunnels in Fig. 1. Fig. 3 shows an electron diffraction pattern taken along
the [010] direction. Some additional weak superstructure spots are observed.
The superstructure spot (indicated by A) is situated at a non-integral
multiple position of the subcell spots along the c*-axis, indicating that
the incommensurate superstructure has a multiplicity of 2.2 x c of the
subcell. The non-integral periodicity can be seen in a high-resolution image
taken along the [010] direction, as shown in Fig. 4. At the thick crystal
regions, some weak dark bands running parallel to the c axis are observed,
in which they have two different widths (2 x c or 2.5 x c) along the c axis.
An average distance between the adjacent dark bands becomes about 2.2 x c,
which is consistent with an optical diffraction pattern (inset in Fig. 4).
One of the possible models for the incommensurate superstructure of the
hexagonal tungsten bronze is proposed in Fig. 5. The superstructure arises
from the local ordering of K ion vacancies located in the tunnels along the
c axis. The vacancies are formed at every fourth or fifth site of K ions. We
call them structures with n = 4 and n = 5. The incommensurate superstructure
results from a mixture of the structure elements with n = 4 and n = 5,
causing the formation of a non-integral periodicity observed presently. It
should be noted in Fig. 4 that the image from a thin region does not show
the superstructure but the image from a thick region does. It seems that
this phenomenon arises from dynamical diffraction effects. This will be
discussed in detail on the basis of the image calculation.
Structure Imaging of Cadmium Telluride
Authors: Yamashita Tom
Published: January 2003

The structure image of a compound semiconductor, cadmium telluride, is
obtained in the 110 projection using high resolution electron microscopy. In
this study, the possibility of identifying individual cadmium and tellurium
atoms in the experimental image is explored. A computer program based on the
Bloch-wave formulation is used for the calculation of the structure images
[1].
The Role of Microscopy in Understanding Atherosclerotic
Lysosomal Lipid Metabolism
Authors: Jerome W. Gray , Yancey Patricia G.
Published: January 2003

Microscopy has played a critical role in first identifying
and then defining the role of lysosomes in formation of
atherosclerotic foam cells. We review the evidence implicating
lysosomal lipid accumulation as a factor in the pathogenesis
of atherosclerosis with reference to the role of microscopy.
In addition, we explore mechanisms by which lysosomal lipid
engorgement occurs. Low density lipoproteins which have become
modified are the major source of lipid for foam cell formation.
These altered lipoproteins are taken into the cell via
receptor-mediated endocytosis and delivered to lysosomes. Under
normal conditions, lipids from these lipoproteins are metabolized
and do not accumulate in lysosomes. In the atherosclerotic foam
cell, this normal metabolism is inhibited so that cholesterol
and cholesteryl esters accumulate in lysosomes. Studies of cultured
cells incubated with modified lipoproteins suggests this abnormal
metabolism occurs in two steps. Initially, hydrolysis of
lipoprotein cholesteryl esters occurs normally, but the resultant
free cholesterol cannot exit the lysosome. Further lysosomal
cholesterol accumulation inhibits hydrolysis, producing a mixture
of cholesterol and cholesteryl esters within swollen lysosomes.
Various lipoprotein modifications can produce this lysosomal
engorgement in vitro and it remains to be seen which
modifications are most important in vivo.
Improved Count Rate and Resolution Performance of a
Thermoelectrically Cooled Si(Li) X-ray Detector
Authors: Barkan S. , Ihrig K. F. , Abott M. B.
Published: January 2003

This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). A laser-scanned confocal light microscope was used to record 3-D images i which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum
nuclear size. The hippocampal data sets were also manually counted independently by five trained observers
using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting.
Topographic Imaging of Chromium-Coated Frozen-Hydrated Cell and Macromolecular Complexes by In-Lens Field Emission Scanning Electron Microscopy
Authors: Apkarian Robert P. , Caran Kevin L. , Robinson Keith A.
Published: January 2003

An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission
electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed
to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information.
High-resolution topographic images at high primary magnification ([eg ]200,000 times) were digitally recorded
with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr
coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed
macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The 'vitreous' nature of bulk
water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present
they would be [el ]2–3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically
difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected
specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and
record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast
cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed.
A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These
experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in
cryofixed cells and cell-free systems.
The Economic Base of Rural Areas: A SAM-Based Analysis of the Western Isles, 1997
Authors: Roberts Deborah
Published: January 2003

A model based on social accounting techniques is used to quantify the relative importance of traditional and nontraditional elements of the economic base of rural areas. Empirical analysis is focused on the Western Isles, Scotland. The results highlight the importance of central government funding of public services. They also indicate that exogenous transfers of income direct to households support 8% of jobs and 7% of factor earnings in the region. It is argued that, in addition to industry structure and input–output linkages, rural economic-base multipliers depend on the demographic profile of the local population and the extent of interdependencies between local production and consumption.
Directed evolution approach to a structural genomics project: Rv2002 from
Mycobacterium
tuberculosis
Authors: Yang Jin Kuk , Park Min S. , Waldo Geoffrey S. , Suh Se Won
Published: January 2003

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the
Mycobacterium tuberculosis
Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in
Escherichia coli
. A triple mutant I6T/V47M/T69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3α, 20β-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD
+
and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140/Tyr-153/Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in
M. tuberculosis
. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.
Caspase 3 activation is essential for neuroprotection in preconditioning
Authors: McLaughlin BethAnn , Hartnett Karen A. , Erhardt Joseph A. , Legos Jeffrey J. , White Ray F. , Barone Frank C. , Aizenman Elias
Published: January 2003

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an
in vivo
model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an
in vitro
model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against
N
-methyl-
d
-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 up-regulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival.
Routes to
S
-nitroso-hemoglobin formation with heme redox and preferential reactivity in the β subunits
Authors: Luchsinger Benjamin P. , Rich Eric N. , Gow Andrew J. , Williams Elizabeth M. , Stamler Jonathan S. , Singel David J.
Published: January 2003

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the α subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the β subunit (cysβ-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of
S
-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the β subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of β subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme–iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.
Correlated terrestrial and marine evidence for global climate changes before mass extinction at the Cretaceous–Paleogene boundary
Authors: Wilf Peter , Johnson Kirk R. , Huber Brian T.
Published: January 2003

Terrestrial climates near the time of the end-Cretaceous mass extinction are poorly known, limiting understanding of environmentally driven changes in biodiversity that occurred before bolide impact. We estimate paleotemperatures for the last ≈1.1 million years of the Cretaceous (≈66.6–65.5 million years ago, Ma) by using fossil plants from North Dakota and employ paleomagnetic stratigraphy to correlate the results to foraminiferal paleoclimatic data from four middle- and high-latitude sites. Both plants and foraminifera indicate warming near 66.0 Ma, a warming peak from ≈65.8 to 65.6 Ma, and cooling near 65.6 Ma, suggesting that these were global climate shifts. The warming peak coincides with the immigration of a thermophilic flora, maximum plant diversity, and the poleward range expansion of thermophilic foraminifera. Plant data indicate the continuation of relatively cool temperatures across the Cretaceous–Paleogene boundary; there is no indication of a major warming immediately after the boundary as previously reported. Our temperature proxies correspond well with recent
p
CO
2
data from paleosol carbonate, suggesting a coupling of
p
CO
2
and temperature. To the extent that biodiversity is correlated with temperature, estimates of the severity of end-Cretaceous extinctions that are based on occurrence data from the warming peak are probably inflated, as we illustrate for North Dakota plants. However, our analysis of climate and facies considerations shows that the effects of bolide impact should be regarded as the most significant contributor to these plant extinctions.
Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system
Authors: Batchelor Eric , Goulian Mark
Published: January 2003

The EnvZ/OmpR system in
Escherichia coli
, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling. Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR. We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation. The model predicts that when EnvZ is much less abundant than OmpR, as is the case in
E. coli
, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ. The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high. To test these predictions, we have perturbed the porin regulatory circuit in
E. coli
by varying the expression levels of EnvZ and OmpR. We have constructed two-color fluorescent reporter strains in which
ompF
and
ompC
transcription can be easily measured in the same culture. Using these strains we have shown that, consistent with the predictions of our model, the transcription of
ompC
and
ompF
is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.
8-Nitroguanosine formation in viral pneumonia and its implication for pathogenesis
Authors: Akaike Takaaki , Okamoto Shinichiro , Sawa Tomohiro , Yoshitake Jun , Tamura Fumio , Ichimori Koji , Miyazaki Kiminori , Sasamoto Kazumi , Maeda Hiroshi
Published: January 2003

For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation
in vivo
. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virus-infected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8- Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOS-deficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS
in vitro
. The present data constitute a demonstration of 8-nitroguanosine formation
in vivo
and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.
Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge
Authors: López-García Purificación , Philippe Hervé , Gail Françoise , Moreira David
Published: January 2003

The diversity and mode of life of microbial eukaryotes in hydrothermal systems is very poorly known. We carried out a molecular survey based on 18S ribosomal RNA genes of eukaryotes present in different hydrothermal niches at the Mid-Atlantic Ridge. These included metal-rich and rare-earth-element-rich hydrothermal sediments of the Rainbow site, fluid–seawater mixing regions, and colonization devices (microcolonizers) containing organic, iron-rich, and porous mineral substrates that were exposed for 15 days to a fluid source. We identified considerable phylogenetic diversity, both at kingdom level and within kinetoplastids and alveolates. None of our sequences affiliates to photosynthesizing lineages, suggesting that we are targeting only autochthonous deep-sea communities. Although sediment harbored most phylogenetic diversity, microcolonizers predominantly contained bodonids and ciliates, indicating that these protists pioneer the colonization process. Given the large variety of divergent lineages detected within the alveolates in deep-sea plankton, hydrothermal sediments, and vents, alveolates seem to dominate the deep ocean in terms of diversity. Compared with data from the Pacific Guaymas basin, some protist lineages seem ubiquitous in hydrothermal areas, whereas others, notably kinetoplastid lineages, very abundant and diverse in our samples, so far have been detected only in Atlantic systems. Unexpectedly, although alvinellid polychaetes are considered endemic of Pacific vents, we detected alvinellid-related sequences at the fluid–seawater interface and in microcolonizers. This finding can boost further studies on deep-sea vent animal biology and biogeography.
Reductive genome evolution in
Buchnera aphidicola
Authors: van Ham Roeland C. H. J. , Kamerbeek Judith , Palacios Carmen , Rausell Carolina , Abascal Federico , Bastolla Ugo , Fernández Jose M. , Jiménez Luis , Postigo Marina , Silva Francisco J. , Tamames Javier , Viguera Enrique , Latorre Amparo , Valencia Alfonso , Morán Federico , Moya Andrés
Published: January 2003

We have sequenced the genome of the intracellular symbiont
Buchnera aphidicola
from the aphid
Baizongia pistacea
. This strain diverged 80–150 million years ago from the common ancestor of two previously sequenced
Buchnera
strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of ≈1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other
Buchnera
genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, ≈200 million years ago. Extensive genome reduction also predates the synchronous diversification of
Buchnera
and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in
Buchnera
, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like
Buchnera
.
Optimal ion channel clustering for intracellular calcium signaling
Authors: Shuai J. W. , Jung P.
Published: January 2003

Ion channels and receptors in the cell membranes and internal membranes are often distributed in discrete clusters. One particularly well-studied example is the distribution of inositol 1,4,5-triphosphate receptors in the plasma membrane that controls the flux of Ca
2+
from the endoplasmic reticulum into the cytosol. By using mathematical modeling, we show that channel clustering can enhance the cell's Ca
2+
signaling capability. Furthermore, we predict optimal signaling cellular capability at cluster sizes and distances that agree with experimentally found values in
Xenopus
oocyte.
Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex
Authors: Wang Haoyong , Chong Shaorong
Published: January 2003

During overexpression of recombinant proteins in
Escherichia coli
, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented
in vivo
by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the
E. coli
integration host factors α and β, and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor β or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein–protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.
Ku is required for telomeric C-rich strand maintenance but not for end-to-end chromosome fusions in
Arabidopsis
Authors: Riha Karel , Shippen Dorothy E.
Published: January 2003

Telomere dysfunction arising from mutations in telomerase or in telomere capping proteins leads to end-to-end chromosome fusions. Paradoxically, the Ku70/80 heterodimer, essential for nonhomologous end-joining double-strand break repair, is also found at telomeres, and in mammals it is required to prevent telomere fusion. Previously, we showed that inactivation of
Ku70
in
Arabidopsis
results in telomere lengthening. Here, we have demonstrated that this telomere elongation is telomerase dependent. Further, we found that the terminal 3′ G overhang was significantly extended in
ku70
mutants and in plants deficient in both Ku70 and the catalytic subunit of telomerase (TERT), implying that Ku is needed for proper maintenance of the telomeric C-rich strand. Consistent with inefficient C-strand maintenance, telomere shortening was accelerated in
ku70 tert
double mutants, and the onset of a terminal sterile phenotype was reached two to three times faster than in
tert
single mutants. Unexpectedly, abundant anaphase bridges were found in terminal plants harboring critically shortened telomeres, indicating that Ku is not required for the formation of end-to-end chromosome fusions in telomerase-deficient
Arabidopsis
. Together, these findings define
Ku70
as a gene in higher eukaryotes required for maintenance of the telomeric C-rich strand and underscore the complexity and diversity of molecular interactions at telomeres.
Global genome repair is required to activate
KIN17
, a UVC-responsive gene involved in DNA replication
Authors: Masson Christel , Menaa Farid , Pinon-Lataillade Ghislaine , Frobert Yveline , Chevillard Sylvie , Radicella J. Pablo , Sarasin Alain , Angulo Jaime F.
Published: January 2003

UV light provokes DNA lesions that interfere with replication and transcription. These lesions may compromise cell viability and usually are removed by nucleotide excision repair (NER). In humans, inactivation of NER is associated with three rare autosomal recessive inherited disorders: xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. The NER earliest step is lesion recognition by a complex formed by XPC and HHR23B proteins. In a subsequent step, XPA protein becomes associated to the repair complex. Here we investigate whether XPA and XPC proteins, involved in global genome repair, may contribute to a signal transduction pathway regulating the response to UVC-induced lesions. We monitored the expression of several UVC-induced genes in cells deficient in either a transduction pathway or mutated on an NER gene. Expression of the
KIN17
gene is induced after UVC irradiation independently of p53 and of activating transcription factor 2. However, in human cells derived from XPA or XPC patients the UVC-induced accumulation of
KIN17
RNA and protein is abolished. Our results indicate that the presence of functional XPA and XPC proteins is essential for the up-regulation of the
KIN17
gene after UVC irradiation. They also show that the integrity of global genome repair is required to trigger
KIN17
gene expression and probably other UVC-responsive genes.
Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles
Authors: Dziennik S. R. , Belcher E. B. , Barker G. A. , DeBergalis M. J. , Fernandez S. E. , Lenhoff A. M.
Published: January 2003

Scanning confocal fluorescence microscopy and multiphoton fluorescence microscopy were used to image the uptake of the protein lysozyme into individual ion exchange chromatography particles in a packed bed in real time. Self-sharpening concentration fronts penetrating into the particles were observed at low salt concentrations in all of the adsorbents studied, but persisted to 100 mM ionic strength only in some materials. In other adsorbents, diffuse profiles were seen at these higher salt concentrations, with the transition region exhibiting a pronounced fluorescence peak at the front at intermediate salt concentrations. These patterns in the uptake profiles are accompanied by significant increases in protein uptake rates that are also seen macroscopically in batch uptake experiments. The fluorescence peak appears to be a concentration overshoot that may develop, in part, from an electrokinetic contribution to transport that also enhances the uptake rate. Further evidence for an electrokinetic origin is that the effect is correlated with high adsorbent surface charge densities. Predictions of a mathematical model incorporating the electrokinetic effect are in qualitative agreement with the observations. These findings indicate that mechanisms other than diffusion contribute to protein transport in oppositely charged porous materials and may be exploited to achieve rapid uptake in process chromatography.
CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells
Authors: Qi Chen-Feng , Martensson Annica , Mattioli Michela , Dalla-Favera Riccardo , Lobanenkov Victor V. , Morse Herbert C.
Published: January 2003

The WEHI 231 B cell lymphoma is used as a model of self-tolerance by clonal deletion because B cell receptor (BCR) ligation results in apoptosis. Two critical events precede cell death: an early rise and fall in expression of MYC and cell-cycle arrest associated with enhanced expression of p21, p27, and p53. CTCF is a transcription factor identified as a repressor of MYC recently shown to cause cell growth inhibition. The present studies demonstrate that BCR ligation of WEHI 231 as well as of normal immature B cells greatly increased expression of CTCF in association with down-regulation of MYC followed by growth arrest and cell death. Conditional expression of CTCF in WEHI 231 mimicked BCR ligation with activated cells showing repressed expression of MYC, enhanced expression of p27, p21, p53, and p19ARF, and inhibition of cell growth and induction of apoptosis. In keeping with a central role for CTCF in control of B cell death, conditional expression of a CTCF antisense construct in WEHI 231 resulted in inhibition of p27, p21, p53, and p19ARFin association with enhanced expression of MYC. Activation of the endogenous CTCF locus by BCR ligation was also mimicked by three other routes to apoptotic death in WEHI 231: inhibition of the phosphoinositide 3-kinase or mTOR/FRAP signaling cascades and treatment with transforming growth factor (TGF)-β. Rapid activation of CTCF by BCR ligation or treatment with TGF-β was suppressed by ligation of CD40. These results demonstrate that CTCF is a common determinant to different pathways of death signaling in immature B cells.
Unique inflammatory RNA profiles of microglia in Creutzfeldt–Jakob disease
Authors: Baker Christopher A. , Manuelidis Laura
Published: January 2003

Previous studies in Creutzfeldt–Jakob disease (CJD) have shown that myeloid cells in the periphery as well as derivative microglial cells in the brain are infectious. Microglia can show an activated phenotype before prion protein (PrP) pathology is detectable in brain, and isolated infectious microglia contain very little PrP. To find whether a set of inflammatory genes are significantly induced or suppressed with infection, we analyzed RNA from isolated microglia with relevant cDNA arrays, and identified ≈30 transcripts not previously examined in any transmissible spongiform encephalopathy. This CJD expression profile contrasted with that of uninfected microglia exposed to prototypic inflammatory stimuli such as lipopolysaccharide and IFN-γ, as well as PrP amyloid. These findings underscore inflammatory pathways evoked by the infectious agent in brain. Transcript profiles unique for CJD microglia and other myeloid cells provide opportunities for more sensitive preclinical diagnoses of infectious and noninfectious neurodegenerative diseases.
Modeling of the inhibitory interaction of phospholamban with the Ca
2+
ATPase
Authors: Toyoshima Chikashi , Asahi Michio , Sugita Yuji , Khanna Reena , Tsuda Takeo , MacLennan David H.
Published: January 2003

The inhibitory interaction of phospholamban (PLN) with the sarco(endo)plasmic reticulum Ca
2+
ATPase isoform 1 (SERCA1a) was modeled on the basis of several constraints which included (
i
) spontaneous formation of SS-bridges between mutants L321C in transmembrane helix 4 (M4) of SERCA1a and N27C in PLN and between V89C (M4) and V49C (PLN); (
ii
) definition of the face of the PLN transmembrane helix that interacts with SERCA; (
iii
) cross-linking between Lys-3 of PLN and Lys-397 and Lys-400 of SERCA2a. The crystal structure of SERCA1a in the absence of Ca
2+
, which binds PLN, was used as the structure into which an atomic model of PLN was built. PLN can fit into a transmembrane groove formed by the juxtaposition of M2, the upper part of M4, M6, and M9. In the SERCA1a structure with bound Ca
2+
, this groove is closed, accounting for the ability of Ca
2+
to disrupt PLN–SERCA interactions. Near the cytoplasmic surface of the bilayer, the PLN helix is disrupted to prevent its collision with M4. The model can be extended into the cytoplasmic domain so that Lys-3 in PLN can be cross-linked with Lys-397 and Lys-400 in SERCA1a with little unwinding of the N-terminal helix of PLN.
Contribution of Fas to diabetes development
Authors: Savinov Alexei Y. , Tcherepanov Andrew , Green E. Allison , Flavell Richard A. , Chervonsky Alexander V.
Published: January 2003

Fas (Tnfrsf6, Apo-1, CD95) is a death receptor involved in apoptosis induced in many cell types. Fas have been shown to be expressed by insulin-producing beta cells in mice and humans. However, the importance of Fas in the development of autoimmune diabetes remains controversial. To further evaluate the importance of Fas in pathogenesis of diabetes, we generated NOD mice (nonobese diabetic mice developing spontaneous autoimmune diabetes) with beta cell-specific expression of a dominant-negative point mutation in a death domain of Fas, known as lprcgor Fascg. Spontaneous diabetes was significantly delayed in NOD mice expressing Fascg, and the effect depended on the expression level of the transgene. However, Fascg-bearing mice were still sensitive to diabetes transferred by splenocytes from overtly diabetic NOD mice. At the same time, Fascgexpression did neutralize the accelerating effect of transgenic Fas-ligand expressed by the same beta cells. Thus, both Fas-dependent and -independent mechanisms are involved in beta cell destruction, but interference with the Fas pathway early in disease development may retard or prevent diabetes progression.
A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor
Authors: Jungbluth Achim A. , Stockert Elisabeth , Huang H. J. Su , Collins Vincent P. , Coplan Keren , Iversen Kristin , Kolb Denise , Johns Terrance J. , Scott Andrew M. , Gullick William J. , Ritter Gerd , Cohen Leonard , Scanlan Matthew J. , Cavanee Webster K. , Old Lloyd J.
Published: January 2003

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with ΔEGFR, a deletion mutation lacking exons 2–7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6
ΔEGFR
(mouse fibroblast line NR6 transfected with ΔEGFR). mAb 806 with selective reactivity for NR6
ΔEGFR
in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-ΔEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with ΔEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without ΔEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to ΔEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that ΔEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.
Self-consistent determination of the transition state for protein folding: Application to a fibronectin type III domain
Authors: Paci Emanuele , Clarke Jane , Steward Annette , Vendruscolo Michele , Karplus Martin
Published: January 2003

We present a general approach in which theory and experiments are combined in an iterative manner to provide a detailed description of the transition state ensemble (TSE) for folding. The method is illustrated by applying it to TNfn3, a fibronectin type III domain protein. In the first iteration, a coarse-grained determination of the TSE is carried out by using a limited set of experimental φ values as constraints in a molecular dynamics sampling simulation. The resulting model of the TSE is used to determine the additional residues whose φ value measurement would provide the most information for refining the TSE. Successive iterations with an increasing number of φ value measurements are carried out until no further changes in the properties of the TSE are detected or there are no additional residues whose φ values can be measured. In the study of TNfn3 three iterations were necessary to achieve self-consistency. A retrospective application of the method can be used to determine the accuracy of the TSE results and to find “key residues” for folding, i.e., those that are most important for the formation of the TSE. The approach reported here is an efficient method for finding the structures that make up the TSEs for protein folding. Its use will improve future efforts for their experimental determination and refinement.