bluuebunny/crossref_metadata_2025 huggingface dataset:
Mass spectroscopic identification of wavelength dependent UV laser photoablation fragments from polymethylmethacrylate
Authors: Estler R. C. , Nogar N. S.
Published: July 2002

Samples of polymethylmethacrylate have been irradiated with laser pulses of 266 nm wavelength along with the wavelengths generated via hydrogen-shifted stimulated Raman scattering. A quadrupole mass spectrometer monitors in real time the photoablation products produced during the irradiation. At wavelengths of 266 nm and above, the products are dominated by monomer, CO2, and CO. At wavelengths below 266 nm, a dramatic change of ablation products is observed, with methyl formate appearing as a major photochemical product.
Initial decomposition of GaAs during rapid thermal annealing
Authors: Haynes T. E. , Chu W. K. , Aselage T. L. , Picraux S. T.
Published: July 2002

A technique has been developed for direct, quantitative measurement of the amount of Ga and As evaporated from uncapped GaAs surfaces during rapid thermal annealing (RTA). The method involves collection of the evaporated molecules on a nearby copper film, followed by compositional analysis of the copper film using 5 MeV Rutherford backscattering. We have measured the rates of evaporation from uncapped GaAs surfaces during RTA in the temperature range 600–725 °C and found them to be in reasonable agreement with rates predicted from available measurements of the equilibrium vapor pressures of Ga and As.
Near-field optical scanning microscopy in reflection
Authors: Fischer U. Ch. , Dürig U. T. , Pohl D. W.
Published: July 2002

The resolution of near-field optical scanning microscopy (NFOS) is determined by the dimensions of the microscopic light source rather than the diffraction limit. To demonstrate NFOS in reflection, intensity changes in the (backward) scattering from a 70–100 nm diam hole in a metal film were recorded while the sample was scanned in close proximity to this aperture. Raster-scan images of a planar metal test pattern yield a resolution comparable to the size of the aperture.
Minority-carrier lifetime in GaAs thin films
Authors: Ahrenkiel R. K. , Dunlavy D. J. , Benner J. , Gale R. P. , McClelland R. W. , Gormley J. V. , King B. D.
Published: July 2002

Double-heterostructure devices of type AlxGa1−xAs/GaAs have been fabricated in thin films grown by the cleavage of lateral epitaxial film for transfer process. The electron lifetime in the p-type GaAs is measured by photoluminescence and found to be 32 ns at the 5×1016 cm−3 doping level. This is the largest reported lifetime for a freestanding GaAs thin film.
HIV-1 escape from a small molecule, CCR5-specific entry inhibitor does not involve CXCR4 use
Authors: Trkola Alexandra , Kuhmann Shawn E. , Strizki Julie M. , Maxwell Elizabeth , Ketas Tom , Morgan Tom , Pugach Pavel , Xu Serena , Wojcik Lisa , Tagat Jayaram , Palani Anandan , Shapiro Sherry , Clader John W. , McCombie Stuart , Reyes Gregory R. , Baroudy Bahige M. , Moore John P.
Published: July 2002

To study HIV-1 escape from a coreceptor antagonist,
the R5 primary isolate CC1/85 was passaged in peripheral blood
mononuclear cells with increasing concentrations of the CCR5-specific
small molecule inhibitor, AD101. By 19 passages, an escape
mutant emerged with a >20,000-fold resistance to AD101. This virus was
cross-resistant to a related inhibitor, SCH-C, and partially
resistant to RANTES but still sensitive to CCR5-specific mAbs. The
resistant phenotype was stable; the mutant virus retained AD101
resistance during nine additional passages of culture in the absence of
inhibitor. Replication of the escape mutant in peripheral blood
mononuclear cells completely depended on CCR5 expression and did not
occur in cells from CCR5-Δ32 homozygous individuals. The escape
mutant was unable to use CXCR4 or any other tested coreceptor to enter
transfected cells. Acquisition of CXCR4 use is not the dominant
in vitro
escape pathway for a small molecule CCR5 entry
inhibitor. Instead, HIV-1 acquires the ability to use CCR5 despite the
inhibitor, first by requiring lower levels of CCR5 for entry and then
probably by using the drug-bound form of the receptor.
A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and
in vivo
Authors: Tran Lorren C. , Kissner Jennifer M. , Westerman Larry E. , Sears Amy E.
Published: July 2002

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG
−
virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After
in vivo
infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C
−
or gG
−
virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG
−
virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.
Inhibition of
Xenopus
oocyte meiotic maturation by catalytically inactive protein kinase A
Authors: Schmitt Anja , Nebreda Angel R.
Published: July 2002

Progesterone induces G
2
-arrested
Xenopus
oocytes to develop into fertilizable eggs in a process called meiotic maturation. Protein kinase A (PKA), the cAMP-dependent protein kinase, has long been known to be a potent inhibitor of meiotic maturation, but little information is available on how PKA functions. We have cloned two
Xenopus
PKA catalytic subunit isoforms, XPKAα and XPKAβ. These proteins are 89% identical and both inhibit progesterone-induced meiotic maturation when overexpressed at low levels, suggesting that PKA activity is tightly regulated in the oocyte. Unexpectedly, catalytically inactive XPKA mutants are able to block progesterone-induced maturation as efficiently as the wild-type active XPKA. These mutants also block meiotic maturation induced by Mos, but are less efficient at inhibiting Cdc25C-induced maturation. Our results indicate that PKA can inhibit meiotic maturation by a novel mechanism, which does not require its kinase activity and is also independent of binding to the PKA regulatory subunits.
Spirochete and protist symbionts of a termite (
Mastotermes electrodominicus
) in Miocene amber
Authors: Wier Andrew , Dolan Michael , Grimaldi David , Guerrero Ricardo , Wagensberg Jorge , Margulis Lynn
Published: July 2002

Extraordinary preservation in amber of the Miocene termite
Mastotermes electrodominicus
has led to the discovery of fossil symbiotic microbes. Spirochete bacteria and wood-digesting protists were identified in the intestinal tissue of the insect. Fossil wood (xylem: developing vessel-element cells, fibers, pit connections), protists (most likely xylophagic amitochondriates), an endospore (probably of the filamentous intestinal bacterium
Arthromitus
=
Bacillus
), and large spirochetes were seen in thin section by light and transmission electron microscopy. The intestinal microbiota of the living termite
Mastotermes darwiniensis,
a genus now restricted to northern Australia, markedly resembles that preserved in amber. This is a direct observation of a 20-million-year-old xylophagus termite fossil microbial community.
Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium
Cylindrospermopsis raciborskii
: An FTIR spectroscopic study
Authors: Várkonyi Zsuzsanna , Masamoto Kazuomori , Debreczeny Mónika , Zsiros Ottó , Ughy Bettina , Gombos Zoltán , Domonkos Ildikó , Farkas Tibor , Wada Hajime , Szalontai Balázs
Published: July 2002

The effects of the growth temperature on the lipids and carotenoids of a filamentous cyanobacterium,
Cylindrospermopsis raciborskii
, were studied., The relative amounts of polyunsaturated glycerolipids and myxoxanthophylls in the thylakoid membranes increased markedly when this cyanobacterium was grown at 25°C instead of 35°C. Fourier transform infrared spectroscopy was used to analyze the low-temperature-induced structural alterations in the thylakoid membranes. Despite the higher amount of unsaturated lipids there, conventional analysis of the ν
sym
CH
2
band (characteristic of the lipid disorder) revealed more tightly arranged fatty-acyl chains for the thylakoids in the cells grown at 25°C as compared with those grown at 35°C. This apparent controversy was resolved by a two-component analysis of the ν
sym
CH
2
band, which demonstrated very rigid, myxoxanthophyll-related lipids in the thylakoid membranes. When this rigid component was excluded from the analysis of the thermotropic responses of the ν
sym
CH
2
bands, the expected higher fatty-acyl disorder was observed for the thylakoids prepared from cells grown at 25°C as compared with those grown at 35°C. Both the carotenoid composition and this rigid component in the thylakoid membranes were only growth temperature-dependent; the intensity of the illuminating light during cultivation had no apparent effect on these parameters. We propose that, besides their well-known protective functions, the polar carotenoids in particular may have structural effects on the thylakoid membranes. These effects should be exerted locally—by forming protective patches, in-membrane barriers of low dynamics—to prevent the access of reactive radicals generated in either enzymatic or photosynthetic processes to sensitive spots of the membranes.
Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: Indefinite linear self-association of bacterial cell division protein FtsZ
Authors: Rivas Germán , Fernández Javier A. , Minton Allen P.
Published: July 2002

Recent measurements of sedimentation equilibrium and
sedimentation velocity have shown that the bacterial cell division
protein FtsZ self-associates to form indefinitely long rod-like linear
aggregates in the presence of GDP and Mg
2+
. In the present
study, the newly developed technique of non-ideal tracer sedimentation
equilibrium was used to measure the effect of high concentrations—up
to 150 g/liter—of each of two inert “crowder” proteins,
cyanmethemoglobin or BSA, on the thermodynamic activity and state of
association of dilute FtsZ under conditions inhibiting
(−Mg
2+
) and promoting (+Mg
2+
) FtsZ
self-association. Analysis of equilibrium gradients of both FtsZ and
crowder proteins indicates that, under the conditions of the present
experiment, FtsZ interacts with each of the two crowder proteins
essentially entirely via steric repulsion, which may be accounted for
quantitatively by a simple model in which hemoglobin, albumin, and
monomeric FtsZ are modeled as effective spherical hard particles, and
each oligomeric species of FtsZ is modeled as an effective hard
spherocylinder. The functional dependence of the sedimentation of FtsZ
on the concentrations of FtsZ and either crowder indicates that, in the
presence of high concentrations of crowder, both the weight-average
degree of FtsZ self-association and the range of FtsZ oligomer sizes
present in significant abundance are increased substantially.
Chemically induced supramolecular reorganization of triblock copolymer assemblies: Trapping of intermediate states via a shell-crosslinking methodology
Authors: Ma Qinggao , Remsen Edward E. , Clark Christopher G. , Kowalewski Tomasz , Wooley Karen L.
Published: July 2002

The mechanism of morphological phase transitions was studied for rod-shaped supramolecular assemblies comprised of a poly(acrylic acid)-
block
-poly(methyl acrylate)-
block
-polystyrene (PAA
90
-
b
-PMA
80
-
b
-PS
100
) triblock copolymer in 33% tetrahydrofuran/water after perturbation by reaction with a positively charged water-soluble carbodiimide. Tetrahydrofuran solvation of the hydrophobic core domain provided the dynamic nature required for the rod-to-sphere phase transition to be complete within 30 min. The intermediate morphologies such as fragmenting rods and pearl-necklace structures were trapped kinetically by the subsequent addition of a diamino crosslinking agent, which underwent covalent crosslinking of the shell layer. Alternatively, shell-crosslinked rod-shaped nanostructures with preserved morphology were obtained by the addition of the crosslinking agent before the addition of the carbodiimide, which allowed for the shell crosslinking to be performed at a faster rate than the morphological reorganization. The formation of robust shell-crosslinked nanostructures provides a methodology by which the morphological evolution processes can be observed, and it allows access to otherwise thermodynamically unstable nanostructures.
A human protein containing multiple types of protease-inhibitory modules
Authors: Trexler Mária , Bányai László , Patthy László
Published: July 2002

By using sensitive homology-search and gene-finding programs,
we have found that a genomic region from the tip of the short arm of
human chromosome 16 (16p13.3) encodes a putative secreted protein
consisting of a domain related to the whey acidic protein (WAP) domain,
a domain homologous with follistatin modules of the Kazal-domain family
(FS module), an immunoglobulin-related domain (Ig domain), two tandem
domains related to Kunitz-type protease inhibitor modules (KU domains),
and a domain belonging to the recently defined NTR-module family (NTR
domain). The gene encoding these
W
AP,
F
S,
I
g,
K
U, and
N
TR modules (hereafter
referred to as the
WFIKKN
gene) is intron-depleted—its
single 1,157-bp intron splits the WAP module. The validity of our gene
prediction was confirmed by sequencing a WFIKKN cDNA cloned from a lung
cDNA library. Studies on the tissue-expression pattern of the
WFIKKN
gene have shown that the gene is expressed
primarily in pancreas, kidney, liver, placenta, and lung. As to the
function of the WFIKKN protein, it is noteworthy that it contains FS,
WAP, and KU modules, i.e., three different module types homologous with
domains frequently involved in inhibition of serine proteases. The
protein also contains an NTR module, a domain type implicated in
inhibition of zinc metalloproteinases of the metzincin family.
On the basis of its intriguing homologies, we suggest that the WFIKKN
protein is a multivalent protease inhibitor that may control the action
of multiple types of serine proteases as well as
metalloproteinase(s).
Association of
Escherichia coli
ribosomes with the inner membrane requires the signal recognition particle receptor but is independent of the signal recognition particle
Authors: Herskovits Anat A. , Bibi Eitan
Published: July 2002

In mammalian cells, as well as
Escherichia
coli
, ribosomes translating membrane proteins interact cotranslationally with translocons in the membrane, and this interaction is essential for proper insertion of nascent polypeptides into the membrane. Both the signal recognition particle (SRP) and its receptor (SR) are required for functional association of ribosomes translating integral membrane proteins with the translocon. Herein, we confirm that membrane targeting of
E. coli
ribosomes requires the prokaryotic SRα homolog FtsY
in vivo
. Surprisingly, however, depletion of the
E. coli
SRP54 homolog (Ffh) has no significant effect on binding of ribosomes to the membrane, although Ffh depletion is detrimental to growth. These and other observations suggest that, in
E. coli
, SRP may operate downstream of SR-mediated targeting of ribosomes to the plasma membrane.
The multiple-race population of the United States: Issues and estimates
Authors: Goldstein Joshua R. , Morning Ann J.
Published: July 2002

This paper presents national estimates of the population likely to identify with more than one race in the 2000 census as a result of a new federal policy allowing multiple racial identification. A large number of race-based public policies—including affirmative action and the redistricting provisions of the Voting Rights Act—may be affected by the shift of some 8–18 million people out of traditional single-race statistical groups. The declines in single-race populations resulting from the new classification procedure are likely to be greater in magnitude than the net undercount in the U.S. census at the center of the controversy over using census sampling. Based on ancestry data in the 1990 census and experimental survey results from the 1995 Current Population Survey, we estimate that 3.1–6.6% of the U.S. population is likely to mark multiple races. Our results are substantially higher than those suggested by previous research and have implications for the coding, reporting, and use of multiple response racial data by government and researchers. The change in racial classification may pose new conundrums for the implementation of race-based public policies, which have faced increasing criticism in recent years.
Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence
Authors: Zhong Liangwei , Arnér Elias S. J. , Holmgren Arne
Published: July 2002

Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys
59
-Val-Asn-Val-Gly-Cys
64
, identical to the active site of GR; the other was a selenenylsulfide formed from Cys
497
-SeCys
498
and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys
497
-SeCys
498
residues of one monomer are adjacent to the Cys
59
and Cys
64
residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys
59
and Cys
64
dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys
498
replaced by Cys having a 100-fold lower
k
cat
for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys
59
-Cys
64
was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.
Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H
2
synthases
Authors: Boutaud Olivier , Aronoff David M. , Richardson Jacob H. , Marnett Lawrence J. , Oates John A.
Published: July 2002

Acetaminophen has antipyretic and analgesic properties yet differs from the nonsteroidal antiinflammatory drugs and inhibitors of prostaglandin H synthase (PGHS)-2 by exhibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminophen inhibits PGHS activity with an IC
50
of 4.3 μM in interleukin (IL)-1α-stimulated human umbilical vein endothelial cells, in contrast with an IC
50
of 1,870 μM for the platelet, with 2 μM arachidonic acid as substrate. This difference is not caused by isoform selectivity, because acetaminophen inhibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored the hypothesis that this difference in cellular responsiveness results from antagonism of the reductant action of acetaminophen on the PGHSs by cellular peroxides. Increasing the peroxide product of the PGHS-cyclooxygenase, prostaglandin G
2
(PGG
2
), by elevating the concentration of either enzyme or substrate reverses the inhibitory action of acetaminophen, as does the addition of PGG
2
itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 μM), a major product of the platelet, completely reverses the action of acetaminophen on PGHS-1. Inhibition of PGHS activity by acetaminophen in human umbilical vein endothelial cells is abrogated by
t
-butyl hydroperoxide. Together these findings support the hypothesis that the clinical action of acetaminophen is mediated by inhibition of PGHS activity, and that hydroperoxide concentration contributes to its cellular selectivity.
Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption
Authors: van't Hof R. J. , Armour K. J. , Smith L. M. , Armour K. E. , Wei X. Q. , Liew F. Y. , Ralston S. H.
Published: July 2002

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studiesin vitroandin vivoshowed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFκB and in NFκB-DNA binding, which were reversed by treatment with the NO donorS-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFκB in osteoclast precursors.
Ultrafast diffraction and structural dynamics: The nature of complex molecules far from equilibrium
Authors: Ruan Chong-Yu , Lobastov Vladimir A. , Srinivasan Ramesh , Goodson Boyd M. , Ihee Hyotcherl , Zewail Ahmed H.
Published: July 2002

Studies of molecular structures at or near their equilibrium configurations have long provided information on their geometry in terms of bond distances and angles. Far-from-equilibrium structures are relatively unknown—especially for complex systems—and generally, neither their dynamics nor their average geometries can be extrapolated from equilibrium values. For such nonequilibrium structures, vibrational amplitudes and bond distances play a central role in phenomena such as energy redistribution and chemical reactivity. Ultrafast electron diffraction, which was developed to study transient molecular structures, provides a direct method for probing the nature of complex molecules far from equilibrium. Here we present our ultrafast electron diffraction observations of transient structures for two cyclic hydrocarbons. At high internal energies of ≈4 eV, these molecules display markedly different behavior. For 1,3,5-cycloheptatriene, excitation results in the formation of hot ground-state structures with bond distances similar to those of the initial structure, but with nearly three times the average vibrational amplitude. Energy is redistributed within 5 ps, but with a negative temperature characterizing the nonequilibrium population. In contrast, the ring-opening reaction of 1,3-cyclohexadiene is shown to result in hot structures with a C—C bond distance of over 1.7 Å, which is 0.2 Å away from any expected equilibrium value. Even up to 400 ps, energy remains trapped in large-amplitude motions comprised of torsion and asymmetric stretching. These studies promise a new direction for studying structural dynamics in nonequilibrium complex systems.
Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a
Authors: Alani Rhoda M. , Young Alison Z. , Shifflett Clinton B.
Published: July 2002

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G
1
-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.
Primary defects in the lens underlie complex anterior segment abnormalities of thePax6heterozygous eye
Authors: Collinson J. Martin , Quinn Jane C. , Buchanan Malcolm A. , Kaufman Matthew H. , Wedden Sarah E. , West John D. , Hill Robert E.
Published: July 2002

We describe lens defects in heterozygous small eye mice, and autonomous deficiencies ofPax6+/−cells in the developing lens ofPax6+/+↔Pax6+/−chimeras. Two separate defects of the lens were identified by analyzing the distribution of heterozygous cells in chimeras:Pax6+/−cells are less readily incorporated into the lens placode than wild type, and those that are incorporated into the lens are not maintained efficiently in the proliferating lens epithelium. The lens of chimeric eyes is, therefore, predominantly wild type from embryonic day 16.5 onwards, whereas heterozygous cells contribute normally to all other eye tissues. Eye size and defects of the iris and cornea are corrected in fetal and adult chimeras with up to 80% mutant cells. Therefore, these aspects of the phenotype may be secondary consequences of primary defects in the lens, which has clinical relevance for the human aniridia (PAX6+/−) phenotype.
Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3
Authors: Roberts Andrew W. , Robb Lorraine , Rakar Steven , Hartley Lynne , Cluse Leonie , Nicola Nicos A. , Metcalf Donald , Hilton Douglas J. , Alexander Warren S.
Published: July 2002

Mice lacking suppressor of cytokine signaling 3 (SOCS3) exhibited embryonic lethality with death occurring between days 11 and 13 of gestation. At this stage, SOCS3
−/−
embryos were slightly smaller than wild type but appeared otherwise normal, and histological analysis failed to detect any anatomical abnormalities responsible for the lethal phenotype. Rather, in all SOCS3
−/−
embryos examined, defects were evident in placental development that would account for their developmental arrest and death. The placental spongiotrophoblast layer was significantly reduced and accompanied by increased numbers of giant trophoblast cells. Delayed branching of the chorioallantois was evident, and, although embryonic blood vessels were present in the labyrinthine layer of SOCS3
−/−
placentas, the network of embryonic vessels and maternal sinuses was poorly developed. Yolk sac erythropoiesis was normal, and, although the SOCS3
−/−
fetal liver was small at day 12.5 of gestation (E12.5), normal frequencies of erythroblasts and hematopoietic progenitor cells, including blast forming unit-erythroid (BFU-E) and, colony forming unit-erythroid (CFU-E) were present at both E11.5 and E12.5. Colony formation for both BFU-E and CFU-E from SOCS3
−/−
mice displayed wild-type quantitative responsiveness to erythropoietin (EPO), in the presence or absence of IL-3 or stem cell factor (SCF). These data suggest that SOCS3 is required for placental development but dispensable for normal hematopoiesis in the mouse embryo.
Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity
Authors: Boder Eric T. , Midelfort Katarina S. , Wittrup K. Dane
Published: July 2002

Single-chain antibody mutants have been evolvedin vitrowith antigen-binding equilibrium dissociation constantKd= 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin–biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 105–107yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling duringin vivoaffinity maturation.
Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors
Authors: Mendez Ana , Burns Marie E. , Sokal Izabela , Dizhoor Alexander M. , Baehr Wolfgang , Palczewski Krzysztof , Baylor Denis A. , Chen Jeannie
Published: July 2002

The retina's photoreceptor cells adjust their sensitivity to allow
photons to be transduced over a wide range of light intensities. One
mechanism thought to participate in sensitivity adjustments is
Ca
2+
regulation of guanylate cyclase (GC) by guanylate
cyclase-activating proteins (GCAPs). We evaluated the contribution of
GCAPs to sensitivity regulation in rods by disrupting their expression
in transgenic mice. The GC activity from GCAPs−/− retinas showed no
Ca
2+
dependence, indicating that Ca
2+
regulation of GCs had indeed been abolished. Flash responses from
dark-adapted GCAPs−/− rods were larger and slower than responses
from wild-type rods. In addition, the incremental flash sensitivity of
GCAPs−/− rods failed to be maintained at wild-type levels in bright
steady light. GCAP2 expressed in GCAPs−/− rods restored maximal
light-induced GC activity but did not restore normal flash response
kinetics. We conclude that GCAPs strongly regulate GC activity in mouse
rods, decreasing the flash sensitivity in darkness and increasing the
incremental flash sensitivity in bright steady light, thereby extending
the rod's operating range.
Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E
2
/EP2 receptor pathway
Authors: Elvin Julia A. , Yan Changning , Matzuk Martin M.
Published: July 2002

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor β superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E
2
(PGE
2
) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE
2
within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE
2
and cyclooxygenase inhibitors on this process. PGE
2
can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE
2
to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE
2
-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte–somatic cell interactions in female reproduction.
Regulation of SR protein localization during development
Authors: Sanford Jeremy R. , Bruzik James P.
Published: July 2002

Ser-Arg-rich (SR) proteins play numerous roles in spliceosome assembly and the regulation of splice-site selection. Whereas considerable attention has focused on the mechanistic details of SR protein activities, little is known concerning how these splicing regulators are controlled by the cell. Here we examined the subcellular localization of precursor mRNA splicing factors during early development of the nematode
Ascaris lumbricoides
. In the early embryo, before major zygotic gene activation, most SR proteins, along with RNA polymerase II, are localized in the cytoplasm. As development proceeds, we observe a significant decrease in the cytoplasmic levels of these factors and a concomitant increase in nuclear localization. In contrast, trimethylguanosine-capped small nuclear ribonucleoproteins are predominantly localized in the nucleus throughout this period. We previously showed that the phosphorylation state and activity of SR proteins are regulated during
A. lumbricoides
embryogenesis. These changes correlate with the onset of precursor mRNA splicing and zygotic transcription. Thus, a coordinate change in the subcellular localization of SR proteins and RNA polymerase II occurs at the transition from reliance on maternally deposited factors to embryonic expression. We propose that before zygotic gene activation, SR proteins and RNA polymerase II are stockpiled in the cytoplasm of early embryos, awaiting signals that lead to their activation.
Multiwavelength anomalous diffraction analysis at the M absorption edges of uranium
Authors: Liu Yee , Ogata Craig M. , Hendrickson Wayne A.
Published: July 2002

The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its M
IV
(3.326 Å) and M
V
(3.490 Å) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f′ = −70
e
, f′′ = 80
e
at the M
IV
edge and f′ = −90
e
, f′′ = 105
e
at the M
V
edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the M
IV
edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-Å Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of
0
ϕ
T
−
0
ϕ
A
for replicated determinations is 32°) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.
Thermodynamics “beyond” local equilibrium
Authors: Vilar J. M. G. , Rubí J. M.
Published: July 2002

Nonequilibrium thermodynamics has shown its applicability in a wide
variety of different situations pertaining to fields such as physics,
chemistry, biology, and engineering. As successful as it is, however,
its current formulation considers only systems close to equilibrium,
those satisfying the so-called local equilibrium hypothesis. Here we
show that diffusion processes that occur far away from equilibrium can
be viewed as at local equilibrium in a space that includes all the
relevant variables in addition to the spatial coordinate. In this way,
nonequilibrium thermodynamics can be used and the difficulties and
ambiguities associated with the lack of a thermodynamic description
disappear. We analyze explicitly the inertial effects in diffusion and
outline how the main ideas can be applied to other situations.
Digital in-line holography for biological applications
Authors: Xu Wenbo , Jericho M. H. , Meinertzhagen I. A. , Kreuzer H. J.
Published: July 2002

Digital in-line holography with numerical reconstruction has
been developed into a new tool, specifically for biological
applications, that routinely achieves both lateral and depth
resolution, at least at the micron level, in three-dimensional imaging.
The experimental and numerical procedures have been incorporated into a
program package with a very fast reconstruction algorithm that is now
capable of real-time reconstruction. This capability is demonstrated
for diverse objects, such as suspension of microspheres and biological
samples (diatom, the head of
Drosophila melanogaster
), and
the advantages are discussed by comparing holographic reconstructions
with images taken by using conventional compound light microscopy.
The endocytic receptor protein LRP also mediates neuronal calcium signaling via
N
-methyl-
d
-aspartate receptors
Authors: Bacskai B. J. , Xia M. Q. , Strickland D. K. , Rebeck G. W. , Hyman B. T.
Published: July 2002

The low density lipoprotein receptor-related protein (LRP) is an
endocytic receptor that is a member of the low density lipoprotein
receptor family. We report that the LRP ligand, activated
α
2
-macroglobulin (α
2
M*), induces robust
calcium influx in cultured primary neurons, but not in nonneuronal
LRP-containing cells in the same culture. The calcium influx is
mediated through
N
-methyl-
d
-aspartate
receptor channels, which explains the neuron specificity of the
response. Microapplication of α
2
M* leads to a localized
response at the site of application that dissipates rapidly, suggesting
that the calcium signal is temporally and spatially discrete. Calcium
influx to α
2
M* is blocked by the physiological LRP
inhibitor, receptor-associated protein. Bivalent antibodies to the
extracellular domain of LRP, but not Fab fragments of the same
antibody, cause calcium influx, indicating that the response is
specific to LRP and may require dimerization of the receptor. Thus, LRP
is an endocytic receptor with a novel signaling role.
Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages
Authors: Sanjabi Shomyseh , Hoffmann Alexander , Liou Hsiou-Chi , Baltimore David , Smale Stephen T.
Published: July 2002

A major challenge in the study of gene regulation by NF-κB/Rel transcription factors is to understand, at the biological and mechanistic levels, the selective functions of individual Rel family members. To study selectivity, we have examined the NF-κB/Rel protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 cell development. In nuclear extracts from lipopolysaccharideactivated macrophages, the predominant Rel dimers capable of binding the IL-12 p40 Rel site were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The two heterodimers bound the site with comparable affinities and exhibited comparable transactivation activities. In striking contrast, p40 mRNA and protein concentrations were reduced dramatically in c-Rel−/−macrophages and only modestly in p65−/−macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel−/−macrophages. These results reveal that a c-Rel-containing complex is an essential and selective activator of p40 transcription, which may reflect unique regulatory mechanisms or biological functions of IL-12. Furthermore, because selectivity was not observedin vitroor in transient transactivation experiments, these findings suggest that an understanding of the selectivity mechanism may require an analysis of the endogenous p40 locus.
A continuous assay for DNA cleavage: The application of “break lights” to enediynes, iron-dependent agents, and nucleases
Authors: Biggins John B. , Prudent James R. , Marshall David J. , Ruppen Mark , Thorson Jon S.
Published: July 2002

Although extensive effort has been applied toward
understanding the mechanism by which enediynes cleave DNA, a continuous
assay for this phenomenon is still lacking. In fact, with the exception
of assays for DNase, continuous assays for most DNA cleavage events are
unavailable. This article describes the application of “molecular
break lights” (a single-stranded oligonucleotide that adopts a
stem-and-loop structure and carries a 5′-fluorescent moiety, a
3′-nonfluorescent quenching moiety, and an appropriate cleavage site
within the stem) to develop the first continuous assay for cleavage of
DNA by enediynes. Furthermore, the generality of this approach is
demonstrated by using the described assay to directly compare the DNA
cleavage by naturally occurring enediynes [calicheamicin and
esperamicin), non-enediyne small molecule agents (bleomycin,
methidiumpropyl–EDTA–Fe(II), and EDTA–Fe(II]), as well as the
restriction endonuclease
Bam
HI. Given the simplicity,
speed, and sensitivity of this approach, the described methodology
could easily be extended to a high throughput format and become a new
method of choice in modern drug discovery to screen for novel
protein-based or small molecule-derived DNA cleavage agents.
Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase
Authors: Unciuleac Mihaela , Boll Matthias
Published: July 2002

Benzoyl-CoA reductase (BCR) from the bacterium
Thauera aromatica
catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e
−
. Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [
14
C]ADP to [
14
C]ATP, which was ≈15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (αβγδ-composition) autophosphorylated its γ-subunit when incubated with [γ-
32
P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.
Underediting of glutamate receptor GluR-B mRNA in malignant gliomas
Authors: Maas Stefan , Patt Stephan , Schrey Michael , Rich Alexander
Published: July 2002

In mammals, RNA editing by site-selective adenosine
deamination regulates key functional properties of neurotransmitter
receptors in the central nervous system. Glutamate receptor subunit B
is nearly 100% edited at one position (the Q/R-site), which is
essential for normal receptor function. Its significance is
apparent from mouse models in which a slightly reduced rate of
Q/R-site editing is associated with early onset epilepsy and
premature death. Here we report that in tissues from malignant human
brain tumors, this editing position of glutamate receptor subunit B is
substantially underedited compared with control tissues. We also
observe alterations in editing and alternative splicing of serotonin
receptor 5-HT
2C
transcripts. These changes correlate with a
decrease in enzymatic activity of the editing enzyme adenosine
deaminase acting on RNA (ADAR) 2, as deduced from analysis of ADAR2
self-editing. Our results suggest a role for RNA editing in tumor
progression and may provide a molecular model explaining the occurrence
of epileptic seizures in association with malignant gliomas.
Crystal structure of a human TATA box-binding protein/TATA element complex.
Authors: Nikolov D B , Chen H , Halay E D , Hoffman A , Roeder R G , Burley S K
Published: July 2002

The TATA box-binding protein (TBP) is required by all three eukaryotic RNA polymerases for correct initiation of transcription of ribosomal, messenger, small nuclear, and transfer RNAs. The cocrystal structure of the C-terminal/core region of human TBP complexed with the TATA element of the adenovirus major late promoter has been determined at 1.9 angstroms resolution. Structural and functional analyses of the protein-DNA complex are presented, with a detailed comparison to our 1.9-angstroms resolution structure of Arabidopsis thaliana TBP2 bound to the same TATA box.