bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier.
Authors: Brustovetsky N , Becker A , Klingenberg M , Bamberg E
Published: July 2002

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.
What determines the folding of the chromatin fiber?
Authors: van Holde K , Zlatanova J
Published: July 2002

In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels. We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription. We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms. On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence.
Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module.
Authors: Plaxco K W , Spitzfaden C , Campbell I D , Dobson C M
Published: July 2002

Fibronectin type III modules contain approximately 90 residues and are an extremely common building block of animal proteins. Despite containing a complex all-beta-sheet topology and eight prolines, the refolding of the 10th type III module of human fibronectin has been found to be very rapid, with native core packing, amide hydrogen bonding, and backbone conformation all recovered within 1 s at 5 degrees C. These observations indicate that this domain can overcome many structural characteristics often thought to slow the folding process.
Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.
Authors: Dunbar C E , Seidel N E , Doren S , Sellers S , Cline A P , Metzger M E , Agricola B A , Donahue R E , Bodine D M
Published: July 2002

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.
The AML1/ETO fusion protein activates transcription of BCL-2
Authors: Klampfer Lidija , Zhang Jin , Zelenetz Andrew O. , Uchida Hideo , Nimer Stephen D.
Published: July 2002

The AML1 gene, located on chromosome 21, is involved in several
distinct chromosomal translocations in human leukemia. In t(8;21) acute
myelogenous leukemia (AML), the AML1 gene is juxtaposed to the ETO gene
located on chromosome 8, generating an AML1/ETO fusion protein. Both
AML1/ETO and the AML1 proteins recognize the same consensus
DNA-binding motif (TGT/CGGT), which is found in the promoters of
several genes involved in hematopoiesis. We found that two myeloid
leukemia cell lines with the t(8;21) translocation, Kasumi and SKNO-1,
have elevated levels of BCL-2 protein compared with other myeloid cell
lines. In addition, we identified a consensus AML1 binding site in the
BCL-2 promoter. Thus far, AML1/ETO has been shown to dominantly
repress its target genes; however, we found that AML1/ETO activates
transcription of the BCL-2 gene in U937 cells. This activation requires
the presence of both the runt homology domain (rhd) and the C-terminal
portion of AML1/ETO. We demonstrated sequence specific binding of
both AML1A and AML1/ETO to the TGTGGT sequence in the BCL-2 promoter
and showed that the AML1 binding site is required for responsiveness to
AML1/ETO. Interestingly, AML1A and AML1B do not modulate the activity
of the BCL-2 promoter. The elevated levels of BCL-2 in cells that
express AML1/ETO may prolong their life span and contribute to the
development of t(8;21) leukemia.
Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.
Authors: Fairbairn L J , Lashford L S , Spooncer E , McDermott R H , Lebens G , Arrand J E , Arrand J R , Bellantuono I , Holt R , Hatton C E , Cooper A , Besley G T , Wraith J E , Anson D S , Hopwood J J , Dexter T M
Published: July 2002

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.
Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.
Authors: García-Colunga J , Miledi R
Published: July 2002

Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds.
A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin
Authors: Brown Leonid S. , Kamikubo Hironari , Zimányi László , Kataoka Mikio , Tokunaga Fumio , Verdegem Peter , Lugtenburg Johan , Lanyi Janos K.
Published: July 2002

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (
i
) the pK
a
values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (
ii
) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (
iii
) preventing rotation of the C
13
—C
14
double bond by replacing the retinal with an all-
trans
locked analogue makes little difference to the Schiff base pK
a
. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.
Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force
Authors: Tuvia Shmuel , Almagor Ada , Bitler Arkady , Levin Shlomo , Korenstein Rafi , Yedgar Saul
Published: July 2002

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (
i
) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (
ii
) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.
Molecular cloning, sequence, expression, and processing of the interleukin 16 precursor
Authors: Baier Michael , Bannert Norbert , Werner Albrecht , Lang Kurt , Kurth Reinhard
Published: July 2002

Interleukin 16 (IL-16) has been shown to function as chemoattractant factor, as a modulator of T-cell activation, and as an inhibitor of immunodeficiency virus replication. The recent identification of inconsistencies in published IL-16 cDNA nucleotide sequences led to the proposal that IL-16 is synthesized in the form of a large precursor protein (pro-IL-16). To identify the true transcriptional start of the IL-16 mRNA rapid amplification of cDNA ends methods were applied. The complete pro-IL-16 cDNA was subsequently molecularly cloned, sequenced, and expressed in COS-7 cells. We report here that pro-IL-16 is most likely synthesized as a 67-kDa protein and is encoded from a major 2.6-kb transcript. Recombinant pro-IL-16 polypeptides are specifically cleaved in lysates of CD8(+) cells, suggesting that the naturally secreted bioactive form of IL-16 is smaller than the originally published 130 amino acids fragment. Moreover, in contrast to other interleukins such as IL-15, IL-16 mRNA expression is almost exclusively limited to lymphatic tissues underlining the potential of IL-16 as an immune regulatory molecule.
The 2.1-Å crystal structure of an archaeal preinitiation complex: TATA-box-binding protein/transcription factor (II)B core/TATA-box
Authors: Kosa Peter F. , Ghosh Gourishankar , DeDecker Brian S. , Sigler Paul B.
Published: July 2002

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-Å crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile
Pyrococcus woesei
. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP–DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.
Transmembrane topology of a CLC chloride channel
Authors: Schmidt-Rose Thomas , Jentsch Thomas J.
Published: July 2002

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure–function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of
in vitro
glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.
Molecular evolution of integrins: Genes encoding integrin β subunits from a coral and a sponge
Authors: Brower Danny L. , Brower Sharon M. , Hayward David C. , Ball Eldon E.
Published: July 2002

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin β subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral β
Cn1
and, especially, the sponge β
Po1
sequences are the most divergent of the “β
1
-class” integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other β subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in β
Cn1
and β
Po1
. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.
Centrosome positioning and directionality of cell movements
Authors: Ueda Masahiro , Gräf Ralph , MacWilliams Harry K. , Schliwa Manfred , Euteneuer Ursula
Published: July 2002

In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell movement: the centrosome is located behind the leading edge, suggesting that it serves as a steering device for directional movement. A logical extension of this suggestion is that a change in the direction of cell movement is preceded by a reorientation, or shift, of the centrosome in the intended direction of movement. We have used a fusion protein of green fluorescent protein (GFP) and γ-tubulin to label the centrosome in migrating amoebae of
Dictyostelium discoideum
, allowing us to determine the relationship of centrosome positioning and the direction of cell movement with high spatial and temporal resolution in living cells. We find that the extension of a new pseudopod in a migrating cell precedes centrosome repositioning. An average of 12 sec elapses between the initiation of pseudopod extension and reorientation of the centrosome. If no reorientation occurs within approximately 30 sec, the pseudopod is retracted. Thus the centrosome does not direct a cell’s migration. However, its repositioning stabilizes a chosen direction of movement, most probably by means of the microtubule system.
Raman spectral evidence for hydration forces between collagen triple helices
Authors: Leikin S. , Parsegian V. A. , Yang W.-H. , Walrafen G. E.
Published: July 2002

Hydration forces are thought to result from the energetic cost of water rearrangement near macromolecular surfaces. Raman spectra, collected on the same collagen samples on which these forces were measured, reveal a continuous change in water hydrogen-bonding structure as a function of separation between collagen triple helices. The varying spectral parameters track the force-distance curve. The energetic cost of water “restructuring,” estimated from the spectra, is consistent with the measured energy of intermolecular interaction. These correlations support the idea that the change in water structure underlies the exponentially varying forces seen in this system at least over the 13–18-Å range of interaxial separations.
Mechanical separation of the complementary strands of DNA
Authors: Essevaz-Roulet B. , Bockelmann U. , Heslot F.
Published: July 2002

We describe the mechanical separation of the two complementary
strands of a single molecule of bacteriophage λ DNA. The 3′ and 5′
extremities on one end of the molecule are pulled progressively apart,
and this leads to the opening of the double helix. The typical forces
along the opening are in the range of 10–15 pN. The separation force
signal is shown to be related to the local GC vs. AT content along the
molecule. Variations of this content on a typical scale of 100–500
bases are presently detected.
Otogelin: A glycoprotein specific to the acellular membranes of the inner ear
Authors: Cohen-Salmon Martine , El-Amraoui Aziz , Leibovici Michel , Petit Christine
Published: July 2002

Efforts to identify the specific components of the mammalian inner ear have been hampered by the small number of neuroepithelial cells and the variety of supporting cells. To circumvent these difficulties, we used a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea. A cDNA encoding a predicted 2910-amino acid protein related to mucin has been isolated. Several lines of evidence indicate, however, that this protein does not undergo the
O-
glycosylation characteristic to mucins. As confirmed by immunocytochemistry and biochemical experiments, this protein is specific to the inner ear. Immunohistofluorescence labeling showed that this protein is a component of all the acellular membranes of the inner ear: i.e., the tectorial membrane of the cochlea, the otoconial and accessory membranes of the utricule and saccule, the cupula of the semicircular canals, and a previously undescribed acellular material covering the otoconia of the saccule. The protein has been named otogelin with reference to its localization. A variety of nonsensory cells located underneath these membranes could be identified as synthesizing otogelin. Finally, this study revealed a maturation process of the tectorial membrane, as evidenced by the progressive organization of otogelin labeling into thick and spaced radial fiber-like structures.
The folding pathway of a protein at high resolution from microseconds to seconds
Authors: Nölting Bengt , Golbik Ralph , Neira José L. , Soler-Gonzalez Andrés S. , Schreiber Gideon , Fersht Alan R.
Published: July 2002

We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the first two of its four α-helices. Φ-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleation–condensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The early stages of the multistate folding of the larger, multimodular, barnase have proved experimentally inaccessible. The folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.
Antigen therapy eliminates T cell inflammation by apoptosis: Effective treatment of experimental autoimmune neuritis with recombinant myelin protein P2
Authors: Weishaupt Andreas , Gold Ralf , Gaupp Stefanie , Giegerich Gerhard , Hartung Hans-Peter , Toyka Klaus V.
Published: July 2002

Exposure of T cells to their specific antigen normally results in proliferation, but in the presence of high and repeatedly administered doses of antigen, T cells may undergo apoptosis. Here we demonstrate that i.v. administration of as little as 100 μg of recombinant P2 protein twice daily completely prevents experimental autoimmune neuritis induced by adoptive transfer of neuritogenic P2-specific T cells or by immunization with the neuritogenic P2–peptide-spanning amino acids 53–78. Antigen treatment started after disease onset markedly ameliorated experimental autoimmune neuritis. The mechanism of action may be through programmed T cell death; a profound increase of the rate of apoptosis was seen in inflammatory foci of peripheral nerves and in the spleen. There was no cytokine switch by our Th1 cells after exposure to their specific antigen, but increased secretion of interferon γ and tumor necrosis factor α was demonstrated. High antigen dose therapy using recombinant, pathogen-free protein may prove useful for the treatment of autoimmune inflammatory disorders of the nervous system.
Characterizing semilocal motions in proteins by NMR relaxation studies
Authors: Fischer Mark W. F. , Zeng Lei , Majumdar Ananya , Zuiderweg Erik R. P.
Published: July 2002

The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of15N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the proteinEscherichia coliflavodoxin.
Poliovirus vaccine vectors elicit antigen-specific cytotoxic T cells and protect mice against lethal challenge with malignant melanoma cells expressing a model antigen
Authors: Mandl Stefanie , Sigal Luis J. , Rock Kenneth L. , Andino Raul
Published: July 2002

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector system to induce protective immunity against diverse pathogens. Replication-competent chimeric viruses can be constructed by inserting foreign antigenic sequences within the poliovirus polyprotein. When inserted sequences are flanked by poliovirus protease recognition sites the recombinant polyprotein is processed to mature and functional viral proteins plus the exogenous antigen. It previously has been shown that poliovirus recombinants can induce antibody responses against the inserted sequences but it is not known whether poliovirus or vaccine vectors derived from it can elicit effective cytotoxic T lymphocyte (CTL) responses. To examine the ability of the recombinant poliovirus to induce CTL responses, a segment of the chicken ovalbumin gene, which includes the H2-Kb-restricted CTL epitope SIINFEKL, was cloned at the junction of the P1 and P2 regions. This recombinant virus replicated with near wild-type efficiency in culture and stably expressed high levels of the ovalbumin antigen. Murine and primate cells infected with the recombinant virus appropriately processed the SIINFEKL epitope and presented it within major histocompatibility complex class I molecules. Inoculation of mice with recombinant poliovirus that expresses ovalbumin elicits an effective specific CTL response. Furthermore, vaccination with these recombinant poliovirus induced protective immunity against challenge with lethal doses of a malignant melanoma cell line expressing ovalbumin.
Human thioredoxin reductase from HeLa cells: Selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin
Authors: Gorlatov Sergey N. , Stadtman Thressa C.
Published: July 2002

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.
Homologous pairing in stretched supercoiled DNA
Authors: Strick T. R. , Croquette V. , Bensimon D.
Published: July 2002

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing
in vivo
. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.
Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus
in vivo
Authors: Lam H. H. D. , Bhardwaj A. , O’Connell M. T. , Hanley D. F. , Traystman R. J. , Sofroniew M. V.
Published: July 2002

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using
in vivo
microdialysis we measured conversion of
l
-[
14
C]arginine to
l
-[
14
C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome
c
) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists
N
-methyl-
d
-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.
Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage
Authors: Schmidt Walter K. , Tam Amy , Fujimura-Kamada Konomi , Michaelis Susan
Published: July 2002

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in
Saccharomyces cerevisiae
. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.
DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription
Authors: Majdalani Nadim , Cunning Christofer , Sledjeski Darren , Elliott Tom , Gottesman Susan
Published: July 2002

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the
trp
transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of
rpoS
messenger RNA, suggesting that pairing of DsrA with the
rpoS
message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.
Limited internal friction in the rate-limiting step of a two-state protein folding reaction
Authors: Plaxco Kevin W. , Baker David
Published: July 2002

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant “internal friction” arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.
Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: An unexpected role for intracellular chloride channels
Authors: Davis-Kaplan Sandra R. , Askwith Candice C. , Bengtzen Andrea C. , Radisky Derek , Kaplan Jerry
Published: July 2002

GEF1
is a gene in
Saccharomyces cerevisiae
, which encodes a putative voltage-regulated chloride channel.
gef1
mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu
+
to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl
−
. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl
−
there is very little Cu-loading at any pH. Cl
−
has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl
−
may also bind to Fet3p and that Cu
+
has an allosteric effect on the binding of Cl
−
to the enzyme. Thus, Cl
−
may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.
Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity
Authors: Koltermann Andre , Kettling Ulrich , Bieschke Jan , Winkler Thorsten , Eigen Manfred
Published: July 2002

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (
Eco
RI,
Bam
HI,
Ssp
I, and
Hin
dIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10
4
to 10
5
samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.
Sequence-specific ligation of DNA using RecA protein
Authors: Ferrin Lance J. , Camerini-Otero R. Daniel
Published: July 2002

A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from
Escherichia coli
to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.
3′ Exoribonucleolytic trimming is a common feature of the maturation of small, stable RNAs in
Escherichia coli
Authors: Li Zhongwei , Pandit Shilpa , Deutscher Murray P.
Published: July 2002

In addition to tRNA and 5S RNA,
Escherichia coli
contains several other small, stable RNA species; these are M1, 10Sa, 6S, and 4.5S RNA. Although these RNAs are initially synthesized as precursor molecules, relatively little is known about their maturation. The data presented here show that 3′ exoribonucleolytic trimming is required for the final maturation of each of these molecules. As found previously with tRNA, but not 5S RNA, any one of a number of exoribonucleases can carry out the trimming reaction
in vivo
, although RNases T and PH are most effective. In their absence, large amounts of immature molecules accumulate for most of the RNAs, and these can be converted to the mature forms
in vitro
by the purified RNases. A model is proposed that identifies a structural feature present in all the small, stable RNAs of
E. coli
, and describes how this structure together with the RNases influences the common mechanism for 3′ maturation.
Cytolytic and IFN-γ-producing activities of γδ T cells in the mouse intestinal epithelium are T cell receptor-β-chain dependent
Authors: Kohyama Masako , Nanno Masanobu , Kawaguchi-Miyashita Mariko , Shimada Shinichiro , Watanabe Mamoru , Hibi Toshifumi , Kaminogawa Schuichi , Ishikawa Hiromichi
Published: July 2002

We analyzed the cytolytic activity of intraepithelial T cells (IEL) isolated from the small intestines of 2- to 3-month-old mutant mice rendered deficient in different gene(s) in which the number of IEL expressing either T cell receptor (TCR)-αβ (αβ-IEL) or TCR-γδ (γδ-IEL) were absent or markedly diminished. When compared with wild-type littermates, cytolytic activity of γδ-IEL was sharply attenuated in TCR-β mutant mice but remained unaltered in TCR-α mutant mice in which a minor population of dull TCR-β+(βdim)-IEL was also present. Cytolytic activity of γδ-IEL was maintained in mice doubly homozygous for β2-microglobulin and transporter associated with antigen processing 1 gene mutations in which a conspicuous decrease was noted in absolute numbers of αβ-IEL. In contrast, both TCR-δ and IL-7 receptor-α gene mutations that lead to lack of γδ-IEL generation did not affect the development or cytolytic activity of the remaining αβ-IEL. The anti-CD3 and anti-TCR-γδ mAb-induced IFN-γ production of γδ-IEL showed the same TCR-α and TCR-β mutation-dependent variability. These results indicate that cytolytic and IFN-γ-producing activities of γδ T cells in mouse intestinal epithelium are TCR-β-chain-dependent.
Heightened sensitivity of a lattice of membrane receptors
Authors: Duke T. A. J. , Bray D.
Published: July 2002

Receptor proteins in both eukaryotic and prokaryotic cells have been found to form two-dimensional clusters in the plasma membrane. In this study, we examine the proposition that such clusters might show coordinated responses because of the spread of conformational states from one receptor to its neighbors. A Monte Carlo simulation was developed in which receptors flipped in probabilistic fashion between an active and an inactive state. Conformational energies depended on (i) ligand binding, (ii) a chemical modification of the receptor conferring adaptation, and (iii) the activity of neighboring receptors. Rate constants were based on data from known biological receptors, especially the bacterial Tar receptor, and on theoretical constraints derived from an analogous Ising model. The simulated system showed a greatly enhanced sensitivity to external signals compared with a corresponding set of uncoupled receptors and was operational over a much wider range of ambient concentrations. These and other properties should make a lattice of conformationally coupled receptors ideally suited to act as a “nose” by which a cell can detect and respond to extracellular stimuli.
Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion
Authors: Patrone Cesare , Andersson Sandra , Korhonen Laura , Lindholm Dan
Published: July 2002

Estrogen is known to influence different functions in brain tissue ranging from neuronal development to plasticity and survival, but the mechanisms involved have not been defined clearly. Previous studies have shown the presence of the two estrogen receptors (ERs), ERα and ERβ, in several brain areas, but less is known about the role of estrogen in the peripheral nervous system. Here we demonstrate that dorsal root ganglion (DRG) neurons express ERα and ERβ during early postnatal development and in culture, and that the ERs localize mainly to neuronal cell nuclei. Studying the role of estrogen in DRG, we observed that low concentrations of 17β-estradiol increased survival of cultured DRG neurons deprived of nerve growth factor. 17β-Estradiol up-regulated the expression of the antiapoptotic molecule Bcl-x without affecting that of Bax, suggesting a mechanism by which the hormone counteracted neuronal death. Antiestrogens abolished the action of 17β-estradiol in the DRG neurons, which demonstrates an involvement of ERs. The results show that estrogen and ERs play an important role in the development and survival of DRG neurons.