bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of inactivation on prion conversion of the
Saccharomyces cerevisiae
Ure2 protein
Authors: Baxa Ulrich , Speransky Vladislav , Steven Alasdair C. , Wickner Reed B.
Published: July 2002

The [URE3] infectious protein (prion) of
Saccharomyces cerevisiae
is a self-propagating amyloid form of Ure2p. The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal “prion” domain is responsible for amyloid filament formation (prion conversion). On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p. We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione
S
-transferase, and green fluorescent protein. All formed amyloid filaments
in vitro
, whose diameters increased with the mass of the appended enzyme. The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct. CD data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all β-sheet. In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures. These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in [URE3] cells by a steric blocking mechanism.
An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity inPten+/−mice
Authors: Podsypanina Katrina , Lee Richard T. , Politis Chris , Hennessy Ian , Crane Allison , Puc Janusz , Neshat Mehran , Wang Hong , Yang Lin , Gibbons Jay , Frost Phil , Dreisbach Valley , Blenis John , Gaciong Zbigniew , Fisher Peter , Sawyers Charles , Hedrick-Ellenson Lora , Parsons Ramon
Published: July 2002

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN+/−mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.
Protein docking along smooth association pathways
Authors: Camacho Carlos J. , Vajda Sandor
Published: July 2002

We propose a docking method that mimics the way proteins bind. The method accounts for the dominant driving forces at the different length scales of the protein binding process, allowing for an efficient selection of a downhill path on the evolving receptor-ligand-free energy landscape. Starting from encounter complexes with as much as 10 Å rms deviation from the native conformation, the method locally samples the six dimensional space of rigid-body receptor-ligand structures subject to a van der Waals constraint. The sampling is initially biased only by the desolvation and electrostatic components of the free energy, which capture the partial affinity of unbound structures that are more than 4 Å away from the native state. Below this threshold, improved discrimination is attained by adding an increasing fraction of the van der Waals energy to the force field. The method, with no free parameters, was tested in eight different sets of independently crystallized receptor-ligand structures consistently predicting bound conformations with the lowest free energies and appropriate stability gap around 2 Å from the native complex. This multistage approach is consistent with the underlying kinetics and internal structure of the free energy funnel to the bound state. Implications for the nature of the protein binding pathways are also discussed.
Subpicosecond conformational dynamics of small peptides probed by two-dimensional vibrational spectroscopy
Authors: Woutersen Sander , Mu Yuguang , Stock Gerhard , Hamm Peter
Published: July 2002

The observation of subpicosecond fluctuations in the conformation
of a small peptide in water is demonstrated. We use an experimental
method that is specifically sensitive to conformational dynamics taking
place on an ultrafast time scale. Complementary molecular-dynamics
simulations confirm that the conformational fluctuations exhibit a
subpicosecond component, the time scale and amplitude of which agree
well with those derived from the experiment.
Regulation of β-catenin transformation by the p300 transcriptional coactivator
Authors: Sun Yongnian , Kolligs Frank T. , Hottiger Michael O. , Mosavin Rashid , Fearon Eric R. , Nabel Gary J.
Published: July 2002

The β-catenin protein plays a critical role in embryonic
development and mature tissue homeostasis through its effects on
E-cadherin-mediated cell adhesion and Wnt-dependent signal
transduction. In colon and other cancers, mutations of β-catenin or
the adenomatous polyposis coli (APC) tumor suppressor appear to
stabilize β-catenin and enhance its interaction with T cell factor
(TCF) or lymphoid enhancer factor (Lef) transcription factors. At
present, a complete picture of the means by which β-catenin's
interactions with TCF/Lef proteins contribute to neoplastic
transformation is lacking. We report that the transcriptional
coactivator p300 interacts with β-catenin
in vitro
and
in vivo
and is critical for β-catenin-mediated
neoplastic transformation. p300 synergistically activates
β-catenin/TCF transcription, and their biochemical association
requires the CH1 domain of p300 and a region of β-catenin that
includes its NH
2
-terminal transactivation domain and the
first two armadillo repeats. Lowering of cellular p300 levels by using
a ribozyme directed against p300 reduced TCF transcriptional activity
and inhibited the neoplastic growth properties of a
β-catenin-transformed rat epithelial cell line and a human colon
carcinoma line with a β-catenin mutation. These findings demonstrate
a critical role for p300 in β-catenin/TCF transcription and in
cancers arising from defects in β-catenin regulation.
Efficient assembly of an HIV-1/MLV Gag-chimeric virus in murine cells
Authors: Chen Benjamin K. , Rousso Itay , Shim Sung , Kim Peter S.
Published: July 2002

In human cells infected by HIV type 1 (HIV-1), the viral Gag
protein directs the assembly of nascent viral particles at the plasma
membrane. In murine cells, HIV-1 Gag fails to reach the plasma membrane
and instead forms nonfunctional intracellular aggregates. The viral
determinants of this species incompatibility are previously undefined.
To address this problem, we replaced a region of HIV-1 Gag known to
direct its localization, the matrix (MA) domain, with functionally
homologous regions from Moloney murine leukemia virus (MLV), a murine
retrovirus. An HIV-1 clone carrying such a chimeric Gag protein,
designated murine HIV (MHIV), assembled more efficiently than
nonchimeric HIV-1 and restored plasma membrane localization of Gag in
murine cells. Increased efficiency of viral assembly in murine cells
was observed from MHIV constructs carrying MLV MA in place of HIV-1 MA.
Efficient processing of the HIV-1 capsid protein from the chimeric Gag
polyprotein and subsequent infectivity of MHIV required the presence of
MLV p12 in addition to MLV MA. These findings strongly suggest that the
HIV-1 MA domain of HIV-1 Gag is responsible for the assembly defect in
mouse cells. Although these MHIV do not recruit native HIV-1 Env
efficiently, they are capable of single-round infection when produced
by high-efficiency transfection of human 293 cells and provided with an
HIV-1 Env lacking its cytoplasmic tail. With further adaptation, this
chimeric MHIV approach may provide the basis for creating an infectious
mouse model for HIV/AIDS.
FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.
Authors: Lorenzi M V , Horii Y , Yamanaka R , Sakaguchi K , Miki T
Published: July 2002

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.
Nonlinear dynamics in ventricular fibrillation.
Authors: Hastings H M , Evans S J , Quan W , Chong M L , Nwasokwa O
Published: July 2002

Electrogram recordings of ventricular fibrillation appear complex and possibly chaotic. However, sequences of beat-to-beat intervals obtained from these recordings are generally short, making it difficult to explicitly demonstrate nonlinear dynamics. Motivated by the work of Sugihara on atmospheric dynamics and the Durbin-Watson test for nonlinearity, we introduce a new statistical test that recovers significant dynamical patterns from smoothed lag plots. This test is used to show highly significant nonlinear dynamics in a stable canine model of ventricular fibrillation.
Interresidue hydrogen bonding in a peptide nucleic acid.RNA heteroduplex.
Authors: Torres R A , Bruce T C
Published: July 2002

Previous molecular mechanics calculations suggest that strands of peptide nucleic acids (PNAs) and complementary oligonucleotides form antiparallel duplexes stabilized by interresidue hydrogen bonds. In the computed structures, the amide carbonyl oxygen nearest the nucleobase (O7') forms an interresidue hydrogen bond with the backbone amide proton of the following residue, (n + 1)H1'. Of the 10 published two dimensional 1H NMR structures of a hexameric PNA.RNA heteroduplex. PNA(GAACTC).r(GAGUUC), 9 exhibit two to five potential interresidue hydrogen bonds. In our minimized average structure, created from the coordinates of these 10 NMR structures, three of the five possible interresidue hydrogen bond sites within the PNA backbone display the carbonyl oxygen (O7') and the amide proton (n + 1)H1' distances and N1'-H1'-(n - 1)O7' angles optimal for hydrogen bond formation. The finding of these interresidue hydrogen bonds supports the results of our previous molecular mechanics calculations.
Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.
Authors: Morris G F , Bischoff J R , Mathews M B
Published: July 2002

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.
Oncogenic potential of the adenovirus E4orf6 protein.
Authors: Moore M , Horikoshi N , Shenk T
Published: July 2002

The group C adenovirus E4orf6 protein has previously been shown to bind to the p53 cellular tumor suppressor protein and block its ability to activate transcription. Here we show that the E4orf6 protein blocks the induction of p53-mediated apoptosis when AT6 cells, which harbor a temperature-sensitive p53, are shifted to the permissive temperature. The E4orf6 protein does not, however, prevent the induction of apoptosis in p53-deficient H1299 cells by treatment with tumor necrosis factor alpha and cycloheximide. The E4orf6 protein also cooperates with the adenovirus E1A protein to transform primary baby rat kidney cells, and it cooperates with the adenovirus E1A plus E1B 19-kDa and E1B 55-kDa proteins to increase the number of baby rat kidney cell transformants and enhance the rate at which they arise. The level of p53 is substantially reduced in transformed cells expressing the E4orf6 protein in comparison to adenovirus transformants lacking it. The E4orf6 gene also accelerates tumor formation when transformed baby rat kidney cells are injected subcutaneously into the nude mouse, and it converts human 293 cells from nontumorigenic to tumorigenic in nude mice. In addition to the well-studied E1A and E1B oncogenes, group C adenoviruses harbor a third oncogene, E4orf6, which functions in some respects similarly to the E1B oncogene.
Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.
Authors: Sharma S , Cantwell M , Kipps T J , Friedmann T
Published: July 2002

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.
A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation
Authors: Joshi Hemalata M. , Tabita F. Robert
Published: July 2002

Photosynthesis, biological nitrogen fixation, and carbon dioxide
assimilation are three fundamental biological processes catalyzed by
photosynthetic bacteria. In the present study, it is shown that mutant
strains of the nonsulfur purple photosynthetic bacteria
Rhodospirillum rubrum
and
Rhodobacter
sphaeroides
, containing a blockage in the primary
CO
2
assimilatory pathway, derepress the synthesis of
components of the nitrogen fixation enzyme complex and abrogate normal
control mechanisms. The absence of the Calvin–Benson–Bassham (CBB)
reductive pentose phosphate CO
2
fixation pathway removes an
important route for the dissipation of excess reducing power. Thus, the
mutant strains develop alternative means to remove these reducing
equivalents, resulting in the synthesis of large amounts of nitrogenase
even in the presence of ammonia. This response is under the control of
a global two-component signal transduction system previously found to
regulate photosystem biosynthesis and the transcription of genes
required for CO
2
fixation through the CBB pathway and
alternative routes. In addition, this two-component system directly
controls the ability of these bacteria to grow under nitrogen-fixing
conditions. These results indicate that there is a molecular link
between the CBB and nitrogen fixation process, allowing the cell to
overcome powerful control mechanisms to remove excess reducing power
generated by photosynthesis and carbon metabolism. Furthermore, these
results suggest that the two-component system integrates the expression
of genes required for the three processes of photosynthesis, nitrogen
fixation, and carbon dioxide fixation.
Interisland and interarchipelago transfer of stone tools in prehistoric Polynesia.
Authors: Weisler M I , Kirch P V
Published: July 2002

Tracing interisland and interarchipelago movements of people and artifacts in prehistoric Polynesia has posed a challenge to archaeologists due to the lack of pottery and obsidian, two materials most readily used in studies of prehistoric trade or exchange. Here we report the application of nondestructive energy-dispersive x-ray fluorescence (EDXRF) analysis to the sourcing of Polynesian artifacts made from basalt, one of the most ubiquitous materials in Polynesian archaeological sites. We have compared excavated and surface-collected basalt adzes and adze flakes from two sites in Samoa (site AS-13-1) and the Cook Islands (site MAN-44), with source basalts from known prehistoric quarries in these archipelagoes. In both cases, we are able to demonstrate the importing of basalt adzes from Tutuila Island, a distance of 100 km to Ofu Island, and of 1600 km to Mangaia Island. These findings are of considerable significance for Polynesian prehistory, as they demonstrate the movement of objects not only between islands in the same group (where communities were culturally and linguistically related) but also between distant island groups. Further applications of EDXRF analysis should greatly aid archaeologists in their efforts to reconstruct ancient trade and exchange networks, not only in Polynesia but also in other regions where basalt was a major material for artifact production.
T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.
Authors: Neophytou P I , Roep B O , Arden S D , Muir E M , Duinkerken G , Kallan A , de Vries R R , Hutton J C
Published: July 2002

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.
Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells.
Authors: Yonemura Y , Ku H , Hirayama F , Souza L M , Ogawa M
Published: July 2002

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self-renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated.
Evidence for a mediator cycle at the initiation of transcription
Authors: Svejstrup Jesper Q. , Li Yang , Fellows Jane , Gnatt Averell , Bjorklund Stefan , Kornberg Roger D.
Published: July 2002

Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.
Streaming potential measurements in αβγ-rat epithelial Na
+
channel in planar lipid bilayers
Authors: Ismailov Iskander I. , Shlyonsky Vadim Gh. , Benos Dale J.
Published: July 2002

Streaming potentials across cloned epithelial Na
+
channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (Δ
P
) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by Δ
P
when the Δπ was directed against Δ
P
. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na
+
] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na
+
ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by Δ
P
can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.
Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions
Authors: Miick Siobhan M. , Fee Richard S. , Millar David P. , Chazin Walter J.
Published: July 2002

Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.
Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between
Salmonella typhimurium
and
Salmonella typhi
Authors: Zahrt Thomas C. , Maloy Stanley
Published: July 2002

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between
Escherichia coli
and
Salmonella typhimurium
and between
S. typhimurium
and
Salmonella typhi
. However, even in mutants defective for mismatch repair the recombination frequencies are 10
2
- to 10
3
-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between
S. typhimurium
and
S. typhi
is less than in transductions between strains of
S. typhimurium
. In homeologous transductions, mutations in the
recD
gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in
mutS recD
double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of
mutS
and
recD
act independently. Because
S. typhimurium
and
S. typhi
are ≈98–99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.
Polyphosphate kinase as a nucleoside diphosphate kinase in
Escherichia coli
and
Pseudomonas aeruginosa
Authors: Kuroda Akio , Kornberg Arthur
Published: July 2002

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or overabundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from
Escherichia coli
and
Pseudomonas aeruginosa
as well as with the purified PPK from
E. coli
; the activity was absent from the membrane fractions obtained from
E. coli
mutants lacking the
ppk
gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2–60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5′ tetraphosphate.
Cloning and characterization of the
Flavobacterium johnsoniae
(
Cytophaga johnsonae
) gliding motility gene,
gldA
Authors: Agarwal Sarika , Hunnicutt David W. , McBride Mark J.
Published: July 2002

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of nonmotile mutants of the gliding bacterium
Flavobacterium johnsoniae
(
Cytophaga johnsonae
) have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nonmotile mutant of
F. johnsoniae
(UW102-09) with a library of wild-type DNA by using the shuttle cosmid pCP17. The complementing plasmid (pCP100) contained an insert of 13 kbp, and restored motility to 4 of 61 independently isolated nonmotile mutants. A 1.3-kbp fragment that encompassed a single ORF,
gldA
, complemented all four mutants. Disruption of the chromosomal copy of
gldA
in wild-type
F. johnsoniae
UW101 eliminated gliding motility. The predicted protein produced by
gldA
has strong sequence similarity to ATP binding cassette transport proteins.
Therapeutic passive vaccination against chronic Lyme disease in mice
Authors: Zhong Weimin , Stehle Thomas , Museteanu Crisan , Siebers Annette , Gern Lise , Kramer Michael , Wallich Reinhard , Simon Markus M.
Published: July 2002

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with
Borrelia burgdorferi
. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 μg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of
B. burgdorferi
resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 μg OspC-specific Abs) were repeatedly given (4× in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.
Local DNA stretching mimics the distortion caused by the TATA box-binding protein
Authors: Lebrun Anne , Shakked Zippora , Lavery Richard
Published: July 2002

X-ray structures of the TATA box-binding protein complexed with its
DNA target show that the nucleic acid is severely bent away from the
protein and also strongly unwound. We have used molecular mechanics and
energy mapping to understand how such an unusual conformation can be
induced. The results show that simple deformation pathways involving
local stretching or unwinding of DNA reproduce many features of the
experimental structure. Notably, kinked junctions with the flanking
B-DNA regions occur without the need for any specific local
interactions with the protein. It is also shown that phosphate
neutralization plays an important role in the formation of the complex.
Osmolyte-driven contraction of a random coil protein
Authors: Qu Youxing , Bolen C. L. , Bolen D. W.
Published: July 2002

The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this “random coil” protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine
N
-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.
Nitric oxide dioxygenase: An enzymic function for flavohemoglobin
Authors: Gardner Paul R. , Gardner Anne M. , Martin Lori A. , Salzman Andrew L.
Published: July 2002

Nitric oxide (NO
•
) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO
•
-resistant mutants of
Escherichia coli
were isolated that rapidly consumed NO
•
. An NO
•
-converting activity was reconstituted in extracts that required NADPH, FAD, and O
2
, was cyanide-sensitive, and produced NO
3
−
. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the
E. coli
flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO
•
. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO
•
. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO
•
detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.
Propagating structure of Alzheimer’s β-amyloid
(10–35)
is parallel β-sheet with residues in exact register
Authors: Benzinger Tammie L. S. , Gregory David M. , Burkoth Timothy S. , Miller-Auer Hélène , Lynn David G. , Botto Robert E. , Meredith Stephen C.
Published: July 2002

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln
15
of one peptide is proximate to Lys
16
of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl
13
C label at Gln
15
, Lys
16
, Leu
17
, or Val
18
were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.
Neuronal polarity: Essential role of protein–lipid complexes in axonal sorting
Authors: Ledesma Maria Dolores , Simons Kai , Dotti Carlos G.
Published: July 2002

The viral glycoprotein hemagglutinin (HA) and the endogenous glycosylphosphatidylinositol-anchored protein Thy-1 are efficiently targeted to the axonal surface of fully polarized hippocampal neurons in culture. Here we have shown that in these cells HA and Thy-1 interact with sphingolipid–cholesterol rafts and are included in detergent-insoluble glycolipid-enriched complexes. Axonal HA and Thy-1, but not two dendritic membrane proteins, resisted extraction to detergents at 4°C. Both HA and Thy-1 became detergent-soluble in neurons with reduced levels of cholesterol or sphingolipids. Missorting of the axonal Thy-1 but not of a dendritic membrane protein occurred in sphingolipid-deprived cells. These results indicate that neurons sort a subset of axolemmal proteins by a mechanism that requires the formation of protein–lipid rafts. The involvement of rafts in axonal membrane sorting may explain the neurological deficits observed in patients with certain types of Niemann–Pick disease.
Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: Dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059
Authors: Mohr Susanne , McCormick Thomas S. , Lapetina Eduardo G.
Published: July 2002

Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donorS-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.
A model for a
umuDC
-dependent prokaryotic DNA damage checkpoint
Authors: Opperman Timothy , Murli Sumati , Smith Bradley T. , Walker Graham C.
Published: July 2002

The products of the
Escherichia coli umuDC
operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals. The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA/single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage. This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD′, the form active in translesion synthesis. Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is
uvr
+
dependent. Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted. After a UV dose of 20 J/m
2
, uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD′ becomes the predominant form, suggesting that the
umuDC
gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions. The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed.
Evidence for microscopic, long-range hydration forces for a hydrophobic amino acid
Authors: Pertsemlidis Alexander , Soper Alan K. , Sorenson Jon M. , Head-Gordon Teresa
Published: July 2002

We have combined neutron solution scattering experiments with molecular dynamics simulation to isolate an excess experimental signal that is caused solely by N-acetyl-leucine-amide (NALA) correlations in aqueous solution. This excess signal contains information about how NALA molecule centers are correlated in water, and we show how these solute–solute correlations might be determined at dilute concentrations in the small angle region. We have tested qualitatively different pair distribution functions for NALA molecule centers—gas, cluster, and aqueous forms of

g
c
(r)

—and have found that the excess experimental signal is adequate enough to rule out gas and cluster pair distribution functions. The aqueous form of

g
c
(r)

that exhibits a solvent-separated minimum, and possibly longer-ranged correlations as well, is not only physically sound but reproduces the experimental data reasonably well. This work demonstrates that important information in the small angle region can be mined to resolve solute–solute correlations, their lengthscales, and thermodynamic consequences even at dilute concentrations. The hydration forces that operate on the microscopic scale of individual amino acid side chains, implied by the small angle scattering data, could have significant effects on the early stages of protein folding, on ligand binding, and on other intermolecular interactions.
Markov Chain Monte Carlo analysis of human Y-chromosome microsatellites provides evidence of biased mutation
Authors: Cooper Gillian , Burroughs Nigel J. , Rand David A. , Rubinsztein David C. , Amos William
Published: July 2002

We describe a Markov Chain Monte Carlo analysis of five human Y- chromosome microsatellite polymorphisms based on samples from five diverse populations. Our analysis provides strong evidence for mutational bias favoring increase in length at all loci. Estimates of population coalescent times and population size from our two largest samples, one African and one European, suggest that the African population is older but smaller and that the English East Anglian population has undergone significant expansion, being larger but younger. We conclude that Markov Chain Monte Carlo analysis of microsatellite haplotypes can uncover information not apparent when the microsatellites are considered independently. Incorporation of population size as a variable should allow us to estimate the timing and magnitude of major historical population trends.
Mobilization of the A-kinase
N-
myristate through an isoform-specific intermolecular switch
Authors: Gangal Milind , Clifford Teresa , Deich Jason , Cheng Xiaodong , Taylor Susan S. , Johnson David A.
Published: July 2002

Although the catalytic (C) subunit of cAMP-dependent protein kinase is
N
-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (R
I
and R
II
) with the
N
-myristylated C subunit affects its ability to target membranes, the effect of
N
-myristylation and the R
I
and R
II
subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of
N
-myristylation and R
II
subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R
II
subunit also increased the conformational flexibility of the C subunit N terminus, the effect of
N
-myristylation and the R
I
and R
II
subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the R
II
subunit, but not the R
I
subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R
II
subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the
N
-myristate. R
II
subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the
N
-myristate and poise the holoenzyme for interaction with membranes.