bluuebunny/crossref_metadata_2025 huggingface dataset:
Two rhodopsins mediate phototaxis to low- and high-intensity light in
Chlamydomonas
reinhardtii
Authors: Sineshchekov Oleg A. , Jung Kwang-Hwan , Spudich John L.
Published: July 2002

We demonstrate that two rhodopsins, identified from cDNA sequences, function as low- and high-light-intensity phototaxis receptors in the eukaryotic alga
Chlamydomonas reinhardtii
. Each of the receptors consists of an ≈300-residue seven-transmembrane helix domain with a retinal-binding pocket homologous to that of archaeal rhodopsins, followed by ≈400 residues of additional membrane-associated portion. The function of the two rhodopsins,
Chlamydomonas
sensory rhodopsins A and B (CSRA and CSRB), as phototaxis receptors is demonstrated by
in vivo
analysis of photoreceptor electrical currents and motility responses in transformants with RNA interference (RNA
i
) directed against each of the rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor currents differ greatly in transformants in accord with the relative amounts of photoreceptor pigments expressed. The data show that CSRA has an absorption maximum near 510 nm and mediates a fast photoreceptor current that saturates at high light intensity. In contrast, CSRB absorbs maximally at 470 nm and generates a slow photoreceptor current saturating at low light intensity. The relative wavelength dependence of CSRA and CSRB activity in producing phototaxis responses matches precisely the wavelength dependence of the CSRA- and CSRB-generated currents, demonstrating that each receptor mediates phototaxis. The saturation of the two photoreceptor currents at different light fluence levels extends the range of light intensity to which the organism can respond. Further, at intensities where both operate, their light signals are integrated at the level of membrane depolarization caused by the two photoreceptor currents.
Structure of the yeast ribonucleotide reductase Y2Y4 heterodimer
Authors: Voegtli Walter C. , Ge Jie , Perlstein Deborah L. , Stubbe JoAnne , Rosenzweig Amy C.
Published: July 2002

The R2 subunits of class I ribonucleotide reductases (RNRs) house a diferric-tyrosyl radical (Y·) cofactor essential for DNA synthesis. In yeast, there are two R2 proteins, Y2 and Y4. Although both Y2 and Y4 are homologous to R2s from other organisms, Y4 lacks three conserved iron-binding residues, and its exact function is unclear. Y4 is required for assembly of the diferric-Y· cofactor in Y2, and the two proteins can form both homodimeric and heterodimeric complexes. The Y2Y4 heterodimer was crystallized from a mixture of the two proteins, and its structure was determined to 2.8 Å resolution. Both Y2 and Y4 are completely α helical and resemble the mouse and
Escherichia coli
R2s in overall fold. Three α helices not observed in the mouse R2 structure are present at the Y2 N terminus, and one extra N-terminal helix is observed in Y4. In addition, one of the eight principal helices in both Y2 and Y4, αD, is shifted significantly from its position in mouse R2. The heterodimer interface is similar to the mouse R2 homodimer interface in size and interacting residues, but loop regions at the interface edges differ. A single metal ion, assigned as Zn(II), occupies the Fe2 position in the Y2 active site. Treatment of the crystals with Fe(II) results in difference electron density consistent with formation of a diiron center. No metal-binding site is observed in Y4. Instead, the residues in the active site region form a hydrogen-bonding network involving an arginine, two glutamic acids, and a water molecule.
Drought- and salt-tolerant plants result from overexpression of the AVP1 H
+
-pump
Authors: Gaxiola Roberto A. , Li Jisheng , Undurraga Soledad , Dang Lien M. , Allen Gethyn J. , Alper Seth L. , Fink Gerald R.
Published: July 2002

Transgenic plants overexpressing the vacuolar
H
+
-pyrophosphatase are much more resistant to high
concentrations of NaCl and to water deprivation than the isogenic
wild-type strains. These transgenic plants accumulate more
Na
+
and K
+
in their leaf tissue than the wild
type. Moreover, direct measurements on isolated vacuolar membrane
vesicles derived from the
AVP1
transgenic plants and
from wild type demonstrate that the vesicles from the transgenic plants
have enhanced cation uptake. The phenotypes of the
AVP1
transgenic plants suggest that increasing the vacuolar proton gradient
results in increased solute accumulation and water retention.
Presumably, sequestration of cations in the vacuole reduces their toxic
effects. Genetically engineered drought- and salt-tolerant plants could
provide an avenue to the reclamation of farmlands lost to
agriculture because of salinity and a lack of rainfall.
Multiple myeloma disrupts the TRANCE/ osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression
Authors: Pearse Roger N. , Sordillo Emilia M. , Yaccoby Shmuel , Wong Brian R. , Liau Deng F. , Colman Neville , Michaeli Joseph , Epstein Joshua , Choi Yongwon
Published: July 2002

Bone destruction, caused by aberrant production and activation of osteoclasts, is a prominent feature of multiple myeloma. We demonstrate that myeloma stimulates osteoclastogenesis by triggering a coordinated increase in the tumor necrosis factor-related activation-induced cytokine (TRANCE) and decrease in its decoy receptor, osteoprotegerin (OPG). Immunohistochemistry andin situhybridization studies of bone marrow specimens indicate thatin vivo, deregulation of the TRANCE–OPG cytokine axis occurs in myeloma, but not in the limited plasma cell disorder monoclonal gammopathy of unknown significance or in nonmyeloma hematologic malignancies. In coculture, myeloma cell lines stimulate expression of TRANCE and inhibit expression of OPG by stromal cells. Osteoclastogenesis, the functional consequence of increased TRANCE expression, is counteracted by addition of a recombinant TRANCE inhibitor, RANK-Fc, to marrow/myeloma cocultures. Myeloma–stroma interaction also has been postulated to support progression of the malignant clone. In the SCID-hu murine model of human myeloma, administration of RANK-Fc both prevents myeloma-induced bone destruction and interferes with myeloma progression. Our data identify TRANCE and OPG as key cytokines whose deregulation promotes bone destruction and supports myeloma growth.
Riverine barriers and the geographic distribution of Amazonian species
Authors: Gascon Claude , Malcolm Jay R. , Patton James L. , da Silva Maria N. F. , Bogart James P. , Lougheed Stephen C. , Peres Carlos A. , Neckel Selvino , Boag Peter T.
Published: July 2002

Rivers have been suggested to have played an important role in
shaping present-day patterns of ecological and genetic variation among
Amazonian species and communities. Recent molecular studies have
provided mixed support for the hypothesis that large lowland Amazonian
rivers have functioned as significant impediments to gene flow among
populations of neotropical species. To date, no study has
systematically evaluated the impact that riverine barriers might have
on structuring whole Amazonian communities. Our analyses of the
phylogeography of frogs and small mammals indicate that a putative
riverine barrier (the Juruá River) does not relate to present-day
patterns of community similarity and species richness. Rather, our
results imply a significant impact of the Andean orogenic axis and
associated thrust-and-fold lowland dynamics in shaping patterns of
biotic diversity along the Juruá. Combined results of this and
other studies significantly weaken the postulated role of rivers as
major drivers of Amazonian diversification.
Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO
2
Authors: Crafts-Brandner Steven J. , Salvucci Michael E.
Published: July 2002

Net photosynthesis (Pn) is inhibited by moderate heat stress. To
elucidate the mechanism of inhibition, we examined the effects of
temperature on gas exchange and ribulose 1,5-bisphosphate
carboxylase/oxygenase (Rubisco) activation in cotton and tobacco
leaves and compared the responses to those of the isolated enzymes.
Depending on the CO
2
concentration, Pn decreased when
temperatures exceeded 35–40°C. This response was inconsistent with
the response predicted from the properties of fully activated Rubisco.
Rubisco deactivated in leaves when temperature was increased and also
in response to high CO
2
or low O
2
. The decrease
in Rubisco activation occurred when leaf temperatures exceeded 35°C,
whereas the activities of isolated activase and Rubisco were highest at
42°C and >50°C, respectively. In the absence of activase, isolated
Rubisco deactivated under catalytic conditions and the rate of
deactivation increased with temperature but not with CO
2
.
The ability of activase to maintain or promote Rubisco activation
in vitro
also decreased with temperature but was not
affected by CO
2
. Increasing the activase/Rubisco ratio
reduced Rubisco deactivation at higher temperatures. The results
indicate that, as temperature increases, the rate of Rubisco
deactivation exceeds the capacity of activase to promote activation.
The decrease in Rubisco activation that occurred in leaves at high
CO
2
was not caused by a faster rate of deactivation, but by
reduced activase activity possibly in response to unfavorable ATP/ADP
ratios. When adjustments were made for changes in activation state, the
kinetic properties of Rubisco predicted the response of Pn at high
temperature and CO
2
.
Expression of
Borrelia burgdorferi
OspC and DbpA is controlled by a RpoN–RpoS regulatory pathway
Authors: Hübner Anette , Yang Xiaofeng , Nolen Dena M. , Popova Taissia G. , Cabello Felipe C. , Norgard Michael V.
Published: July 2002

RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria. To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another. Herein, using a combined strategy of gene disruption and genetic complementation targeting
rpoN
and
rpoS
in
Borrelia burgdorferi
strain 297, we describe a regulatory network for
B. burgdorferi
. In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of
B. burgdorferi
. Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of
B. burgdorferi
's parasitic strategy, host range, and virulence expression.
Microfluidic tectonics: A comprehensive construction platform for microfluidic systems
Authors: Beebe David J. , Moore Jeffrey S. , Yu Qing , Liu Robin H. , Kraft Mary L. , Jo Byung-Ho , Devadoss Chelladurai
Published: July 2002

A microfluidic platform for the construction of microscale
components and autonomous systems is presented. The platform combines
liquid-phase photopolymerization, lithography, and laminar flow to
allow the creation of complex and autonomous microfluidic systems. The
fabrication of channels, actuators, valves, sensors, and systems is
demonstrated. Construction times can be as short as 10 min, providing
ultrarapid prototyping of microfluidic systems.
Disruption ofPPT1orPPT2causes neuronal ceroid lipofuscinosis in knockout mice
Authors: Gupta Praveena , Soyombo Abigail A. , Atashband Armita , Wisniewski Krystyna E. , Shelton John M. , Richardson James A. , Hammer Robert E. , Hofmann Sandra L.
Published: July 2002

PPT1andPPT2encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create “knockout” mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a “clasping” phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.
Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-
l
- carnitine and/or
R
-α-lipoic acid
Authors: Liu Jiankang , Killilea David W. , Ames Bruce N.
Published: July 2002

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-
l
-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor
R
-α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels.
Ex vivo
oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity.
In vitro
oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However,
in vitro
treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.
DNA analysis and diagnostics on oligonucleotide microchips.
Authors: Yershov G , Barsky V , Belgovskiy A , Kirillov E , Kreindlin E , Ivanov I , Parinov S , Guschin D , Drobishev A , Dubiley S , Mirzabekov A
Published: July 2002

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.
Extremely sensitive, background-free gene detection using binary probes and beta replicase.
Authors: Tyagi S , Landegren U , Tazi M , Lizardi P M , Kramer F R
Published: July 2002

We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range of target concentrations and it employs a universal format that can detect any infectious agent.
Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice.
Authors: Dymecki S M
Published: July 2002

Site-specific recombinases are being developed as tools for "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. Here it is demonstrated that Flp recombinase can effectively mediate site-specific excisional recombination in mouse embryonic stem cells, in differentiating embryonal carcinoma cells, and in transgenic mice. Broad Flp expression is compatible with normal development, suggesting that Flp can be used to catalyze recombination in most cell types. These properties indicate that Flp can be exploited to make prescribed alterations in the mouse genome.
A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.
Authors: Mitra A , Higgins D W , Langenberg W G , Nie H , Sengupta D N , Silverman R H
Published: July 2002

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.
A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.
Authors: Babcook J S , Leslie K B , Olsen O A , Salmon R A , Schrader J W
Published: July 2002

We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans.
Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.
Authors: Rejto P A , Verkhivker G M
Published: July 2002

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.
The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome.
Authors: Wu S H , Lagarias J C
Published: July 2002

Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.
Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract.
Authors: Connell I , Agace W , Klemm P , Schembri M , Mărild S , Svanborg C
Published: July 2002

Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces and inflammatory cells in vitro, but their contribution to virulence has not been defined. This study presents evidence that type 1 fimbriae increase the virulence of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory response to infection. In a clinical study, we observed that disease severity was greater in children infected with E. coli O1:K1:H7 isolates expressing type 1 fimbriae than in those infected with type 1 negative isolates of the same serotype. The E. coli O1:K1:H7 isolates had the same electrophoretic type, were hemolysin-negative, expressed P fimbriae, and carried the fim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:K1:H7 isolates survived in higher numbers, and induced a greater neutrophil influx into the urine, than O1:K1:H7 type 1-negative isolates. To confirm a role of type 1 fimbriae, a fimH null mutant (CN1016) was constructed from an O1:K1:H7 type 1-positive parent. E. coli CN1016 had reduced survival and inflammatogenicity in the mouse urinary tract infection model. E. coli CN1016 reconstituted with type 1 fimbriae (E. coli CN1018) had restored virulence similar to that of the wild-type parent strain. These results show that type 1 fimbriae in the genetic background of a uropathogenic strain contribute to the pathogenesis of E. coli in the urinary tract.
Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones.
Authors: Vitale M , Sivori S , Pende D , Augugliaro R , Di Donato C , Amoroso A , Malnati M , Bottino C , Moretta L , Moretta A
Published: July 2002

Natural killer (NK) cells express clonally distributed receptors for different groups of HLA class I alleles. The Z27 monoclonal antibody described in this study recognizes a p70 receptor specific for HLA-B alleles belonging to the Bw4 supertypic specificity. Single amino acid substitutions in the peptide-binding groove of HLA-B2705 molecules influenced the recognition by some, but not all, p7O/Z27+ clones. This suggests the existence of a limited polymorphism within the p7O family of receptors. The pattern of reactivity of monoclonal antibody Z27 revealed that Bw4-specific receptors may be expressed alone or in combination with different (GL183 and/or EB6) p58 molecules. Analysis of NK clones coexpressing p58 and p7O receptors allowed us to demonstrate that the two molecules represent physically and functionally independent receptors. The expression of p7O molecules either alone or in combination with EB6 molecules provided the molecular basis for understanding the cytolytic pattern of two previously defined groups of "alloreactive" NK cell clones ("group 3" and "group 5").
Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain.
Authors: Kopan R , Schroeter E H , Weintraub H , Nye J S
Published: July 2002

Previous studies imply that the intracellular domain of Notch1 must translocate to the nucleus for its activity. In this study, we demonstrate that a mNotch1 mutant protein that lacks its extracellular domain but retains its membrane-spanning region becomes proteolytically processed on its intracellular surface and, as a result, the activated intracellular domain (mNotchIC) is released and can move to the nucleus. Proteolytic cleavage at an intracellular site is blocked by protease inhibitors. Intracellular cleavage is not seen in cells transfected with an inactive variant, which includes the extracellular lin-Notch-glp repeats. Collectively, the studies presented here support the model that mNotch1 is proteolytically processed and the cleavage product is translocated to the nucleus for mNotch1 signal transduction.
In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.
Authors: Sera T , Schultz P G
Published: July 2002

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.
Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.
Authors: Hinterdorfer P , Baumgartner W , Gruber H J , Schilcher K , Schindler H
Published: July 2002

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.
Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.
Authors: Boudreau N , Werb Z , Bissell M J
Published: July 2002

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.
Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells
Authors: Lin Mark Mu-Quan , Zhu Minghua , Scharff Matthew D.
Published: July 2002

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisenin vivoreveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.
Estimation of the DNA sequence discriminatory ability of hairpin-linked lexitropsins
Authors: Walker Wynn L. , Landaw Elliot M. , Dickerson Richard E. , Goodsell David S.
Published: July 2002

Three- and four-ring polyamides containing
N
-methylimidazole and
N
-methylpyrrole, and their hairpin-linked derivatives, bind side-by-side in the minor groove of DNA in a sequence-specific manner. The sequences recognized by side-by-side molecules are dependent on the pairings of the polyamide rings to the bases. In this study we report a mathematical model for estimating the free energies of binding for γ-aminobutyric acid-linked polyamides to 5- and 6-bp DNA sequences. The model parameters are calibrated by a least-squares fit to 35 experimental binding constants. The model performs well in cross-validation experiments and the parameters are consistent with previously proposed empirical rules of polyamide–DNA binding. We apply the model to the design of targeted polyamides, evaluating the ability of the proposed polyamides to bind to a DNA sequence of interest while minimizing binding to the remaining DNA sequences.
Cleft palate and decreased brain γ-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase
Authors: Asada Hideo , Kawamura Yuuki , Maruyama Kei , Kume Hideaki , Ding Ria-Go , Kanbara Nobuko , Kuzume Hiroko , Sanbo Makoto , Yagi Takeshi , Obata Kunihiko
Published: July 2002

In addition to its role as an inhibitory neurotransmitter, γ-aminobutyric acid (GABA) is presumed to be involved in the development and plasticity of the nervous system. GABA is synthesized by glutamic acid decarboxylase (GAD), but the respective roles of its two isoforms (GAD65 and 67) have not been determined. The selective elimination of each GAD isoform by gene targeting is expected to clarify these issues. Recently we have produced
GAD65
−/− mice and demonstrated that lack of GAD65 does not change brain GABA contents or animal behavior, except for a slight increase in susceptibility to seizures. Here we report the production of
GAD67
−/− mice. These mice were born at the expected frequency but died of severe cleft palate during the first morning after birth. GAD activities and GABA contents were reduced to 20% and 7%, respectively, in the cerebral cortex of the newborn
GAD67
−/− mice. Their brain, however, did not show any discernible defects. Previous pharmacological and genetic investigations have suggested the involvement of GABA in palate formation, but this is the first demonstration of a role for GAD67-derived GABA in the development of nonneural tissue.
Antigen organization regulates cluster formation and induction of cytotoxic T lymphocytes by helper T cell subsets
Authors: Stuhler Gernot , Schlossman Stuart F.
Published: July 2002

Generation of effector cytotoxic T lymphocytes (CTLs) is a process tightly governed by regulatory helper T (Th) cells. The nature of cellular interactions as well as the precise role of distinct Th cell subsets involved in efficient CTL activation remains elusive. Employingin vitrocultures for primary induction of human, peptide-specific CTL, a strict requirement for Th cells and linkage of epitopes for helper and CTLs on the surface of antigen presenting cells was found, suggesting a three cell type cluster as minimal immune regulatory entity. Cognate and antigen-driven interactions of T cells were neither essential nor sufficient to override the need for linked epitopes. Within the three cell type cluster complex, keyhole limpit hemocyanin or tetanus toxoid-reactive Th cells promoted generation of MAGE-3- or HIV-gag-specific CTL. Both type 1 and type 2 Th cells were recruited and induced by CTL. Interleukin 2 and interferon γ were essential in early stages, and interleukin 4 was utilized in later stages, of CTL maturation. Synergistic effects of CD45RA+and CD45RO+Th cells were found. The data reported here suggest a critical link between the innate and adaptive immune system in the initiation process of cytolytic immune responses and offers the basis for efficient vaccine strategies.
On the coefficients of the characteristic series of the
U
-operator
Authors: Coleman Robert F.
Published: July 2002

A conceptual proof is given of the fact that the coefficients of
the characteristic series of the
U
-operator acting on
families of overconvegent modular forms lie in the Iwasawa algebra.
Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver
Authors: Shimomura Iichiro , Bashmakov Yuriy , Shimano Hitoshi , Horton Jay D. , Goldstein Joseph L. , Brown Michael S.
Published: July 2002

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH
2
-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH
2
-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.
Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells
Authors: García-Hernández B. , Castellanos A. , López A. , Orfao A. , Sánchez-García I.
Published: July 2002

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human
CD5
cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by
de novo
methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection
in vitro
of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.
Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology
Authors: Sturchler-Pierrat Christine , Abramowski Dorothee , Duke Mairead , Wiederhold Karl-Heinz , Mistl Claudia , Rothacher Sabin , Ledermann Birgit , Bürki Kurt , Frey Peter , Paganetti Paolo A. , Waridel Caroline , Calhoun Michael E. , Jucker Mathias , Probst Alphonse , Staufenbiel Matthias , Sommer Bernd
Published: July 2002

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid β (Aβ) peptide, the major constituent of AD plaques. We expressed human APP
751
containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes Aβ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of Aβ in the pathogenesis of the disease.
Peroxynitrite rapidly permeates phospholipid membranes
Authors: Marla Sudhakar S. , Lee Jinbo , Groves John T.
Published: July 2002

Peroxynitrite (ONOO
−
) is a potent oxidant implicated in a number of pathophysiological processes. The activity of ONOO
−
is related to its accessibility to biological targets before its spontaneous decomposition (
t
1/2
≈ 1 s at pH 7.4, 37°C). Using model phospholipid vesicular systems and manganese porphyrins as reporter molecules, we demonstrated that ONOO
−
freely crosses phospholipid membranes. The calculated permeability coefficient for ONOO
−
is ≈8.0 × 10
−4
cm⋅s
−1
, which compares well with that of water and is ≈400 times greater than that of superoxide. We suggest that ONOO
−
is a significant biological effector molecule not only because of its reactivity but also because of its high diffusibility.
Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities
Authors: Sago Haruhiko , Carlson Elaine J. , Smith Desmond J. , Kilbridge Joshua , Rubin Edward M. , Mobley William C. , Epstein Charles J. , Huang Ting-Ting
Published: July 2002

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called “Down syndrome region” of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from
Sod1
to
Mx1
, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.
A new satellite technology for tracking the movements of Atlantic bluefin tuna
Authors: Block Barbara A. , Dewar Heidi , Farwell Charles , Prince Eric D.
Published: July 2002

The movements of Atlantic bluefin tuna (
Thunnus thynnus thynnus
) have captured the interest of scientists and fishers since the time of Aristotle. This tuna is unique among bony fish for maintaining elevated body temperatures (21°C above ambient) and attaining large size (up to 750 kg). We describe here the use of a pop-off satellite tag, for investigating the Atlantic-wide movements and potential stock overlap of western and eastern Atlantic bluefin tuna. The tag also archives data on water temperatures. The objectives of this study were to evaluate the effectiveness of the technology, study the movements of Atlantic bluefin tuna, examine their thermal niche, and assess survivorship of tagged fish. The pop-off satellite technology provides data independent of commercial fisheries that, when deployed in sufficient quantity, should permit a critical test of the stock structure hypotheses for Atlantic bluefin tuna.