bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonlinear-dynamical arrhythmia control in humans
Authors: Christini David J. , Stein Kenneth M. , Markowitz Steven M. , Mittal Suneet , Slotwiner David J. , Scheiner Marc A. , Iwai Sei , Lerman Bruce B.
Published: July 2002

Nonlinear-dynamical control techniques, also known as chaos
control, have been used with great success to control a wide range of
physical systems. Such techniques have been used to control the
behavior of
in vitro
excitable biological tissue,
suggesting their potential for clinical utility. However, the
feasibility of using such techniques to control physiological processes
has not been demonstrated in humans. Here we show that
nonlinear-dynamical control can modulate human cardiac
electrophysiological dynamics by rapidly stabilizing an unstable target
rhythm. Specifically, in 52/54 control attempts in five patients, we
successfully terminated pacing-induced period-2 atrioventricular-nodal
conduction alternans by stabilizing the underlying unstable
steady-state conduction. This proof-of-concept demonstration shows that
nonlinear-dynamical control techniques are clinically feasible and
provides a foundation for developing such techniques for more complex
forms of clinical arrhythmia.
Construction of hybrid peptide synthetases by module and domain fusions
Authors: Mootz Henning D. , Schwarzer Dirk , Marahiel Mohamed A.
Published: July 2002

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly
in vitro
. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide
d
Phe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating
d
Phe, the predicted tripeptides
d
Phe-Pro-Orn and
d
Phe-Pro-Leu were obtained at rates of 0.15 min
-1
and 2.1 min
-1
. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.
Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure
Authors: Holtwick Rita , Gotthardt Michael , Skryabin Boris , Steinmetz Martin , Potthast Regine , Zetsche Bernd , Hammer Robert E. , Herz Joachim , Kuhn Michaela
Published: July 2002

Atrial natriuretic peptide (ANP) is an important regulator of arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve the inhibition of the sympathetic and renin-angiotensin-aldosterone (RAA) systems, increased diuresis/natriuresis, vasodilation, and enhanced vascular permeability. In particular, the contribution of the direct vasodilating effect of ANP to the hypotensive actions remains controversial, because variable levels of the ANP receptor, guanylyl cyclase A (GC-A), are expressed in different vascular beds. The objective of our study was to determine whether a selective deletion of GC-A in vascular smooth muscle would affect the hypotensive actions of ANP. We first created a mutant allele of mouse GC-A by flanking a required exon with loxP sequences. Crossing floxed GC-A with SM22-Cre transgene mice expressing Cre recombinase in smooth muscle cells (SMC) resulted in mice in which vascular GC-A mRNA expression was reduced by ≈80%. Accordingly, the relaxing effects of ANP on isolated vessels from these mice were abolished; despite this fact, chronic arterial blood pressure of awake SMC GC-A KO mice was normal. Infusion of ANP caused immediate decreases in blood pressure in floxed GC-A but not in SMC GC-A knockout mice. Furthermore, acute vascular volume expansion, which causes release of cardiac ANP, did not affect resting blood pressure of floxed GC-A mice, but rapidly and significantly increased blood pressure of SMC GC-A knockout mice. We conclude that vascular GC-A is dispensable in the chronic and critical in the acute moderation of arterial blood pressure by ANP.
Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-β antibody indb/dbdiabetic mice
Authors: Ziyadeh Fuad N. , Hoffman Brenda B. , Han Dong Cheol , Iglesias-de la Cruz M. Carmen , Hong Soon Won , Isono Motohide , Chen Sheldon , McGowan Tracy A. , Sharma Kumar
Published: July 2002

Emerging evidence suggests that transforming growth factor-β (TGF-β) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-β antibody (αT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-β system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of αT prevents renal insufficiency and glomerulosclerosis in thedb/dbmouse, a model of type 2 diabetes that develops overt nephropathy. Diabeticdb/dbmice and nondiabeticdb/mlittermates were treated intraperitoneally with αT or control IgG, 300 μg three times per week for 8 wk. Treatment with αT, but not with IgG, significantly decreased the plasma TGF-β1 concentration without decreasing the plasma glucose concentration. The IgG-treateddb/dbmice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding α1(IV) collagen and fibronectin. On the other hand, treatment with αT completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix indb/dbmice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by αT treatment. We conclude that chronic inhibition of the biologic actions of TGF-β with a neutralizing monoclonal antibody indb/dbmice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.
Stepping rotation of F
1
-ATPase visualized through angle-resolved single-fluorophore imaging
Authors: Adachi Kengo , Yasuda Ryohei , Noji Hiroyuki , Itoh Hiroyasu , Harada Yoshie , Yoshida Masasuke , Kinosita Kazuhiko
Published: July 2002

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F
1
-ATPase revealed that the subunit rotates in the molecule in discrete 120° steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120° stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.
In vitro
and
in vivo
studies of a VEGF
121
/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors
Authors: Veenendaal Liesbeth M. , Jin Hangqing , Ran Sophia , Cheung Lawrence , Navone Nora , Marks John W. , Waltenberger Johannes , Thorpe Philip , Rosenblum Michael G.
Published: July 2002

Vascular endothelial growth factor (VEGF) plays a key role in the growth and metastasis of solid tumors. We generated a fusion protein containing VEGF
121
linked by a flexible G
4
S tether to the toxin gelonin (rGel) and expressed this as a soluble protein in bacteria. Purified VEGF
121
/rGel migrated as an 84-kDa homodimer under nonreducing conditions. VEGF
121
/rGel bound to purified, immobilized Flk-1, and the binding was competed by VEGF
121
. Both VEGF
121
/rGel and VEGF
121
stimulated cellular kinase insert domain receptor (KDR) phosphorylation. The VEGF
121
/rGel fusion construct was highly cytotoxic to endothelial cells overexpressing the KDR/Flk-1 receptor. The IC
50
of the construct on dividing endothelial cells expressing 10
5
or more KDR/Flk-1 receptors per cell was 0.5–1 nM, as compared with 300 nM for rGel itself. Dividing endothelial cells overexpressing KDR were approximately 60-fold more sensitive to VEGF
121
/rGel than were nondividing cells. Endothelial cells overexpressing FLT-1 were not sensitive to the fusion protein. Human melanoma (A-375) or human prostate (PC-3) xenografts treated with the fusion construct demonstrated a reduction in tumor volume to 16% of untreated controls. The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed. These studies demonstrate the successful use of VEGF
121
/rGel fusion construct for the targeted destruction of tumor vasculature
in vivo.
3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: Implications for diabetic nephropathy
Authors: Danesh Farhad R. , Sadeghi Mehran M. , Amro Nail , Philips Carrie , Zeng Lixia , Lin Sun , Sahai Atul , Kanwar Yashpal S.
Published: July 2002

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, also known as statins, are lipid-lowering agents widely used in the prevention of coronary heart disease. Recent experimental and clinical data, however, indicate that the overall benefits of statin therapy may exceed its cholesterol-lowering properties. We postulate that statins may ameliorate the detrimental effects of high glucose (HG)-induced proliferation of mesangial cells (MCs), a feature of early stages of diabetic nephropathy, by preventing Rho isoprenylation. Rat MCs cultured in HG milieu were treated with and without simvastatin, an HMG-CoA reductase inhibitor. Simvastatin inhibited HG-induced MC proliferation as measured by [
3
H]thymidine incorporation. This inhibitory effect was reversed with geranylgeranyl pyrophosphate, an isoprenoid intermediate of the cholesterol biosynthetic pathway. At the cell-cycle level, the HG-induced proliferation of MCs was associated with a decrease in cyclin dependent kinase (CDK) inhibitor p21 protein expression accompanied by an increase in CDK4 and CDK2 kinase activities. Simvastatin reversed the down-regulation of p21 protein expression and decreased CDK4 and CDK2 kinase activities. Exposure of MCs to HG was associated with an increase in membrane-associated Ras and Rho GTPase protein expression. Cotreatment of MCs with simvastatin reversed HG-induced Ras and Rho membrane translocation. Immunofluorescence microscopy revealed that the overexpression of the dominant-negative RhoA led to a significant increase in p21 expression. Our data suggest that simvastatin represses the HG-induced Rho GTPase/p21 signaling in glomerular MCs. Thus, this study provides a molecular basis for the use of statins, independently of their cholesterol-lowering effect, in early stages of diabetic nephropathy.
The generation of oscillations in networks of electrically coupled cells
Authors: Loewenstein Y. , Yarom Y. , Sompolinsky H.
Published: July 2002

In several biological systems, the electrical coupling of nonoscillating cells generates synchronized membrane potential oscillations. Because the isolated cell is nonoscillating and electrical coupling tends to equalize the membrane potentials of the coupled cells, the mechanism underlying these oscillations is unclear. Here we present a dynamic mechanism by which the electrical coupling of identical nonoscillating cells can generate synchronous membrane potential oscillations. We demonstrate this mechanism by constructing a biologically feasible model of electrically coupled cells, characterized by an excitable membrane and calcium dynamics. We show that strong electrical coupling in this network generates multiple oscillatory states with different spatio-temporal patterns and discuss their possible role in the cooperative computations performed by the system.
Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice
Authors: Ju Haisong , Gros Robert , You Xiaomang , Tsang Sarah , Husain Mansoor , Rabinovitch Marlene
Published: July 2002

We cloned a rat vascular chymase (RVCH) from smooth muscle cells (SMCs) that converts angiotensin I to II and is up-regulated in SMC from spontaneously hypertensive vs. normotensive rats. To determine whether increased activity of RVCH is sufficient to cause hypertension, transgenic mice were generated with targeted conditional expression of RVCH to SMC, with the use of the tetracycline-controlled transactivator (tTA). We confirmed conditional expression of RVCH by mRNA, protein, and chymase activity in the absence, but not in the presence, of dietary doxycycline. The systolic blood pressure (mmHg), measured by carotid artery cannulation at 10–12 weeks of age, was higher in tTA+/RVCH+ mice than in nonbinary transgenic littermates (136 ± 4 vs. 109 ± 3) (
P
< 0.05), as were the diastolic and mean pressures. Hypertension was completely reversed by doxycycline, suggesting a causal link with chymase expression. Medial thickening of mesenteric arteries from tTA+/RVCH+ mice vs. littermates (0.82 ± 0.1 vs. 0.42 ± 0.02) (
P
< 0.05) was associated with increased SMC proliferation, as judged by positive immunoreactivity, with the use of an antibody to the proliferating cell nuclear antigen. These structural changes were prevented by doxycycline. Perfusion myography of mesenteric arteries from tTA+/RVCH+ mice also revealed increased vasoconstriction in response to phenylephrine and impaired metacholine-induced vasodilatation when compared with littermate controls or with the doxycyline-treated group. Our studies suggest that up-regulation of this vascular chymase is sufficient to cause a hypertensive arteriopathy, and that RVCH may be a candidate gene and a therapeutic target in patients with high blood pressure.
Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface
Authors: Heino Sanna , Lusa Sari , Somerharju Pentti , Ehnholm Christian , Olkkonen Vesa M. , Ikonen Elina
Published: July 2002

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.
Functional MRI at 1.5 tesla: A comparison of the blood oxygenation level-dependent signal and electrophysiology
Authors: Disbrow Elizabeth A. , Slutsky Daniel A. , Roberts Timothy P. L. , Krubitzer Leah A.
Published: July 2002

How well does the functional MRI (fMRI) signal reflect underlying electrophysiology? Despite the ubiquity of the technique, this question has yet to be adequately answered. Therefore, we have compared cortical maps generated based on the indirect blood oxygenation level-dependent signal of fMRI with maps from microelectrode recording techniques, which directly measure neural activity. Identical somatosensory stimuli were used in both sets of experiments in the same anesthetized macaque monkeys. Our results demonstrate that fMRI can be used to determine the topographic organization of cortical fields with 55% concordance to electrophysiological maps. The variance in the location of fMRI activation was greatest in the plane perpendicular to local vessels. An appreciation of the limitations of fMRI improves our ability to use it effectively to study cortical organization.
T6BP, a TRAF6-interacting protein involved in IL-1 signaling
Authors: Ling Lei , Goeddel David V.
Published: July 2002

We report the identification of a TRAF-interacting protein, T6BP, that specifically associates with TRAF6. This interaction occurs between the coiled-coil region of T6BP and the N-terminal ring finger and zinc finger domains of TRAF6. IL-1, but not tumor necrosis factor, induces TRAF6–T6BP complex formation in a ligand-dependent manner. Formation of the TRAF6–T6BP complex depends on the presence of the IL-1 receptor-associated kinase (IRAK). After IL-1 stimulation, TRAF6 can exist in two separate complexes, TRAF6–IRAK or TRAF6–T6BP, but IRAK is not present in TRAF6–T6BP complexes. T6BP does not seem to play a direct role in activation of IκB kinases or Jun N-terminal kinase.
GIGANTEA is a nuclear protein involved in phytochrome signaling in
Arabidopsis
Authors: Huq Enamul , Tepperman James M. , Quail Peter H.
Published: July 2002

In a genetic screen of available T-DNA-mutagenized
Arabidopsis
populations for loci potentially involved in phytochrome (phy) signaling, we identified a mutant that displayed reduced seedling deetiolation under continuous red light, but little if any change in responsiveness to continuous far-red light. This behavior suggests disruption of phyB, but not phyA signaling. We have cloned the mutant locus by using the T-DNA insertion and found that the disrupted gene is identical to the recently described
GIGANTEA
(
GI
) gene identified as being involved in control of flowering time. The encoded GI polypeptide has no sequence similarity to any known proteins in the database. However, by using β−glucuronidase-GI and green fluorescent protein-GI fusion constructs, we have shown that GI is constitutively targeted to the nucleus in transient transfection assays. Optical sectioning by using the green fluorescent protein-GI fusion protein showed green fluorescence throughout the nucleoplasm. Thus, contrary to previous computer-based predictions that GI would be an integral plasmamembrane-localized polypeptide, the data here indicate that it is a nucleoplasmically localized protein. This result is consistent with the proposed role in phyB signaling, given recent evidence that early phy signaling events are nuclear localized.
Multiple benefits of multiple mating in guppies
Authors: Evans J. P. , Magurran A. E.
Published: July 2002

The rewards of promiscuity for males are undisputed. But why should a female mate promiscuously, particularly when her partners offer no resources other than sperm and increase her chances of succumbing to predation or disease? This question has been hotly debated but at present remains largely unresolved [Jennions, M. D. & Petrie, M. (2000)
Biol. Rev.
75, 21–64]. One possibility is that females exploit postcopulatory mechanisms, such as sperm competition, to increase both the quality and quantity of their offspring. In this paper, we use the Trinidadian guppy, a species with a resource-free mating system, to test the hypothesis that females gain multiple benefits from multiple mating. Our results indicate that multiply mated females secure substantive advantages: They have shorter gestation times and larger broods, and they produce offspring with better developed schooling abilities and escape responses than their singly mated counterparts.
Conversion of monomeric protein L to an obligate dimer by computational protein design
Authors: Kuhlman Brian , O'Neill Jason W. , Kim David E. , Zhang Kam Y. J. , Baker David
Published: July 2002

Protein L consists of a single α-helix packed on a four-stranded β-sheet formed by two symmetrically opposed β-hairpins. We use a computer-based protein design procedure to stabilize a domain-swapped dimer of protein L in which the second β-turn straightens and the C-terminal strand inserts into the β-sheet of the partner. The designed obligate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of ≈700 pM, which is comparable to the dissociation constant of many naturally occurring protein dimers. The structure of the dimer has been determined by x-ray crystallography and is close to the
in silico
model.
Replication-dependent early meiotic requirement for Spo11 and Rad50
Authors: Merino Sandra T. , Cummings W. Jason , Acharya Sonia N. , Zolan Miriam E.
Published: July 2002

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in
Saccharomyces cerevisiae
to initiate meiotic recombination. In both
S. cerevisiae
and the basidiomycete
Coprinus cinereus
,
spo11
and
rad50
mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in
C. cinereus spo11
and
rad50
mutants if premeiotic DNA replication is prevented. Double mutants were constructed with
spo11–1
or
rad50–4
and another mutant,
spo22–1
, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in
C. cinereus
, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.
Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities
Authors: Avbelj Franc , Luo Peizhi , Baldwin Robert L.
Published: July 2002

The alanine helix provides a model system for studying the
energetics of interaction between water and the helical peptide group,
a possible major factor in the energetics of protein folding. Helix
formation is enthalpy-driven (−1.0 kcal/mol per residue).
Experimental transfer data (vapor phase to aqueous) for amides give the
enthalpy of interaction with water of the amide group as ≈−11.5
kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed
for the gas phase by quantum mechanics, is −4.9 kcal/mol. These
numbers give an enthalpy deficit for helix formation of −7.6
kcal/mol. To study this problem, we calculate the electrostatic
solvation free energy (ESF) of the peptide groups in the helical and
β-strand conformations, by using the
delphi
program and
parse
parameter set. Experimental data show that the ESF
values of amides are almost entirely enthalpic. Two key results are: in
the β-strand conformation, the ESF value of an interior alanine
peptide group is −7.9 kcal/mol, substantially less than that of
N
-methylacetamide (−12.2 kcal/mol), and the helical
peptide group is solvated with an ESF of −2.5 kcal/mol. These
results reduce the enthalpy deficit to −1.5 kcal/mol, and
desolvation of peptide groups through partial burial in the random coil
may account for the remainder. Mutant peptides in the helical
conformation show ESF differences among nonpolar amino acids that
are comparable to observed helix propensity differences, but
the ESF differences in the random coil conformation still must be
subtracted.
BCR/ABL inhibition by an escort/phosphatase fusion protein
Authors: Lim Young-Mi , Wong Stephane , Lau Gary , Witte Owen N. , Colicelli John
Published: July 2002

Cellular transformation by the BCR/ABL oncogene depends on
the ABL-encoded tyrosine kinase activity. To block BCR/ABL function,
we created a unique tyrosine phosphatase by fusing the catalytic domain
of SHP1 (SHP1c) to the ABL binding domain (ABD) of RIN1, an established
binding partner and substrate for c-ABL and BCR/ABL. This fusion
construct (ABD/SHP1c) binds to BCR/ABL in cells and functions as an
active phosphatase. ABD/SHP1c effectively suppressed BCR/ABL
function as judged by reductions in transformation of fibroblast cells,
growth factor independence of hematopoietic cell lines, and
proliferation of primary bone marrow cells. In addition, the
leukemogenic properties of BCR/ABL in a murine model system were
blocked by coexpression of ABD/SHP1c. Both the “escort”
function provided by ABD and the inhibitor function provided by the
phosphatase of SHP1c were necessary for effective BCR/ABL
interference. Expression of ABD/SHP1c also reversed the transformed
phenotype of K562, a human leukemia-derived cell line. These results
have direct implications for leukemia therapeutics and suggest an
approach to block aberrant signal transduction in other pathologies
through the use of appropriately designed escort/inhibitors.
Nuclear envelope proteomics: Novel integral membrane proteins of the inner nuclear membrane
Authors: Dreger Mathias , Bengtsson Luiza , Schöneberg Torsten , Otto Henning , Hucho Ferdinand
Published: July 2002

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to
Caenorhabditis elegans
Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.
Presenilin, Notch, and the genesis and treatment of Alzheimer's disease
Authors: Selkoe Dennis J.
Published: July 2002

Elucidation of the proteolytic processing of the amyloid
β-protein precursor (APP) has revealed that one of the two proteases
(γ-secretase) that cleave APP to release amyloid β-protein
(Aβ) is likely to be presenilin. Presenilin also mediates the
γ-secretase-like cleavage of Notch receptors to enable signaling by
their cytoplasmic domains. Therefore, APP and Notch may be the
first identified substrates of a unique intramembranous aspartyl
protease that has presenilin as its active-site component. In view of
the evidence for a central role of cerebral build-up of Aβ in the
pathogenesis of Alzheimer's disease, this disorder appears to have
arisen in the human population as a late-life consequence of the
conservation of a critical developmental pathway.
Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B
2
)-binding protein and in glucose oxidase enzyme
Authors: Zhong Dongping , Zewail Ahmed H.
Published: July 2002

Flavoproteins can function as hydrophobic sites for vitamin B
2
(riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: ≈100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)/tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a charge-separated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase.
Calmodulin priming: Nuclear translocation of a calmodulin complex and the memory of prior neuronal activity
Authors: Mermelstein Paul G. , Deisseroth Karl , Dasgupta Neela , Isaksen Ann L. , Tsien Richard W.
Published: July 2002

The neuronal nucleus plays a vital role in information
processing, but whether it supports computational functions such as
paired-pulse facilitation, comparable to synapses, is unclear.
Ca
2+
-dependent movement of calmodulin (CaM) to
the nucleus is highly responsive to Ca
2+
entry through
L-type channels and promotes activation of the transcription factor
CREB (cAMP-responsive element binding protein) through phosphorylation
by CaM-sensitive kinases. We characterized key features of this CaM
translocation and its possible role in facilitation of nuclear
signaling. Nuclear CaM was elevated within 15 s of stimulus onset,
preceding the first signs of CREB phosphorylation in hippocampal
pyramidal neurons. Depolarization-induced elevation of nuclear CaM also
was observed in cerebellar granule cells, neocortical neurons, and
dentate gyrus granule cells. Nuclear translocation of CaM was not
blocked by disruption of actin filaments or microtubules, or by
emptying endoplasmic reticulum Ca
2+
stores with
thapsigargin. Translocation of fluorescently tagged CaM was prevented
by fusing it with the Ca
2+
/CaM binding peptide M13,
suggesting that nuclear CaM accumulation depends on association with
endogenous Ca
2+
/CaM binding proteins. To
determine whether increased nuclear [CaM] might influence subsequent
nuclear signal processing, we compared responses to two consecutive
depolarizing stimuli. After a weak “priming” stimulus that caused
CaM translocation, CREB phosphorylation caused by a subsequent stimulus
was significantly faster, more sensitive to Ca
2+
elevation,
and less specifically dependent on Ca
2+
influx through
L-type channels. CaM translocation not only supports rapid signaling to
the nucleus, but also could provide a “memory” for facilitatory
effects of repeated neural activity, seen in altered phosphorylated
CREB dynamics and Ca
2+
channel dependence.
PERK mediates cell-cycle exit during the mammalian unfolded protein response
Authors: Brewer Joseph W. , Diehl J. Alan
Published: July 2002

The accumulation of unfolded proteins in the endoplasmic reticulum
(ER) triggers the
u
nfolded
p
rotein
r
esponse (UPR)-signaling pathway. The UPR coordinates
the induction of ER chaperones with decreased protein synthesis and
growth arrest in the G
1
phase of the cell cycle. Three ER
transmembrane protein kinases (Ire1α, Ire1β, and PERK) have been
implicated as proximal effectors of the mammalian UPR. We now
demonstrate that activation of PERK signals the loss of cyclin D1
during the UPR, culminating in cell-cycle arrest. Overexpression of
wild-type PERK inhibited cyclin D1 synthesis in the absence of ER
stress, thereby inducing a G
1
phase arrest. PERK expression
was associated with increased phosphorylation of the translation
elongation initiation factor 2α (eIF2α), an event previously shown
to block cyclin D1 translation. Conversely, a truncated form of PERK
lacking its kinase domain acted as a dominant negative when
overexpressed in cells, attenuating both cyclin D1 loss and cell-cycle
arrest during the UPR without compromising induction of ER chaperones.
These data demonstrate that PERK serves as a critical effector of
UPR-induced growth arrest, linking stress in the ER to control of
cell-cycle progression.
Metal ion cooperativity in ribozyme cleavage of RNA
Authors: Brännvall Mathias , Kirsebom Leif A.
Published: July 2002

Combinations of chemical and genetic approaches were used to study the function of divalent metal ions in cleavage of RNA by the ribozyme RNase P RNA. We show that different divalent metal ions have differential effects on cleavage site recognition and rescue of cleavage activity by mixing divalent metal ions that do not promote cleavage by themselves. We conclude that efficient and correct cleavage is the result of cooperativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex. Complementation of a mutant RNase P RNA phenotype as a result of divalent metal ion replacement is demonstrated also. This finding together with other data indicate that one of the metal ions involved in this cooperativity is positioned near the cleavage site. The possibility that the Mg
2+
/Ca
2+
ratio might regulate the activity of biocatalysts that depend on RNA for activity is discussed.
Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth
Authors: Serrano Antonio L. , Murgia Marta , Pallafacchina Giorgia , Calabria Elisa , Coniglio Patrizia , Lømo Terje , Schiaffino Stefano
Published: July 2002

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin
in vivo
has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.
Identification and characterization of a mitochondrial thioredoxin system in plants
Authors: Laloi Christophe , Rayapuram Naganand , Chartier Yvette , Grienenberger Jean-Michel , Bonnard Géraldine , Meyer Yves
Published: July 2002

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria.
Arabidopsis thaliana
genome sequencing has revealed numerous thioredoxin genes among which we have identified
AtTRX-o1
, a gene encoding a thioredoxin with a potential mitochondrial transit peptide.
AtTRX-o1
and a second gene,
AtTRX-o2
, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized
AtNTRA
, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of
A. thaliana
subcellular and submitochondrial fractions and
in vitro
import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in
Escherichia coli
; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.
SNARE proteins contribute to calcium cooperativity of synaptic transmission
Authors: Stewart Bryan A. , Mohtashami Mahmood , Trimble William S. , Boulianne Gabrielle L.
Published: July 2002

A hallmark of calcium-triggered synaptic transmission is the
cooperative relationship between calcium and the amount of transmitter
released. This relationship is thought to be important for improving
the efficiency of synaptic vesicle exocytosis. Although it is generally
held that cooperativity arises from the interaction of multiple calcium
ions with a single calcium-sensing molecule, the precise molecular
basis of this phenomenon is not known. The SNARE proteins are known to
be critical for synaptic vesicle exocytosis. We therefore tested for a
contribution of SNARE proteins to cooperativity by genetically reducing
the levels of syntaxin IA and neuronal-synaptobrevin in
Drosophila
. Surprisingly, we found that reducing these
SNARE proteins also reduced Ca
2+
cooperativity. Thus, SNARE
proteins are important for determining the cooperative relationship
between calcium and synaptic transmission.
Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA fromTaxusand functional expression inEscherichia coli
Authors: Walker Kevin , Croteau Rodney
Published: July 2002

A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated fromTaxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-inducedTaxuscells, from which several full-length acyltransferases were obtained and individually expressed inEscherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC,1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0,Kmvalues of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position.
Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice
Authors: Roberts Charles W. M. , Galusha Shelly A. , McMenamin Máirín E. , Fletcher Christopher D. M. , Orkin Stuart H.
Published: July 2002

Malignant rhabdoid tumor (MRT) is an aggressive, highly lethal
cancer of young children. Tumors occur in various locations, including
kidney, brain, and soft tissues. Despite intensive therapy, 80% of
affected children die, often within 1 year of diagnosis. The majority
of MRT samples and cell lines have sustained biallelic inactivating
mutations of the
hSNF5
(integrase interactor 1) gene,
suggesting that hSNF5 may act as a tumor suppressor. We sought to
examine the role of Snf5 in development and cancer in a murine model.
Here we report that Snf5 is widely expressed during embryogenesis with
focal areas of high-level expression in the mandibular portion of the
first branchial arch and central nervous system. Homozygous knockout of
Snf5 results in embryonic lethality by embryonic day 7, whereas
heterozygous mice are born at the expected frequency and appear normal.
However, beginning as early as 5 weeks of age, heterozygous mice
develop tumors consistent with MRT. The majority of tumors arise in
soft tissues derived from the first branchial arch. Our findings
constitute persuasive genetic evidence that Snf5, a core member of the
Swi/Snf chromatin-remodeling complex, functions as a tumor suppressor
gene, and, moreover, Snf5 heterozygotes provide a murine model of this
lethal pediatric cancer.
Recombination and mutation during long-term gastric colonization by
Helicobacter pylori
: Estimates of clock rates, recombination size, and minimal age
Authors: Falush Daniel , Kraft Christian , Taylor Nancy S. , Correa Pelayo , Fox James G. , Achtman Mark , Suerbaum Sebastian
Published: July 2002

The bacterium
Helicobacter pylori
colonizes the
gastric mucosa of half of the human population, resulting in chronic
gastritis, ulcers, and cancer. We sequenced ten gene fragments from
pairs of strains isolated sequentially at a mean interval of 1.8 years
from 26 individuals. Several isolates had acquired small mosaic
segments from other
H. pylori
or point mutations. The
maximal mutation rate, the import size, and the frequency of
recombination were calculated by using a Bayesian model. The
calculations indicate that the last common ancestor of
H.
pylori
existed at least 2,500–11,000 years ago. Imported
mosaics have a median size of 417 bp, much smaller than for other
bacteria, and recombination occurs frequently (60 imports spanning
25,000 bp per genome per year). Thus, the panmictic population
structure of
H. pylori
results from very frequent
recombination during mixed colonization by unrelated strains.
The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis
Authors: Sørensen Jakob B. , Matti Ulf , Wei Shun-Hui , Nehring Ralf B. , Voets Thomas , Ashery Uri , Binz Thomas , Neher Erwin , Rettig Jens
Published: July 2002

Synchronous neurotransmission depends on the tight coupling between Ca
2+
influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble
N
-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.
Motexafin gadolinium (Gd-Tex) selectively induces apoptosis in HIV-1 infected CD4+ T helper cells
Authors: Perez Omar D. , Nolan Garry P. , Magda Darren , Miller Richard A. , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: July 2002

Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular oxidative stress, selectively induces apoptosis in HIV-1-infected CD4
+
T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected
in vitro
with HIV-1. This selective induction of apoptosis, which we detect by FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis marker expression, is abrogated by the glutathione precursor,
N
-acetyl-
l
-cysteine. Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected cells in the culture. These findings suggest that Gd-Tex may have therapeutic utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected cells in an infected host.