bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Peptide Recognized by Five Melanoma-Specific Human Cytotoxic T Cell Lines
Authors: Cox Andrea L. , Skipper Jonathan , Chen Ye , Henderson Robert A. , Darrow Timothy L. , Shabanowitz Jeffrey , Engelhard Victor H. , Hunt Donald F. , Slingluff Craig L.
Published: October 2006

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.
Homozygous Human TAP Peptide Transporter Mutation in HLA Class I Deficiency
Authors: de la Salle Henri , Hanau Daniel , Fricker Dominique , Urlacher Arlette , Kelly Adrian , Salamero Jean , Powis Stephen H. , Donato Lionel , Bausinger Huguette , Laforet Michel , Jeras Matjaz , Spehner Danièle , Bieber Thomas , Falkenrodt Annie , Cazenave Jean-Pierre , Trowsdale John , Tongio Marie-Marthe
Published: October 2006

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8
+
αβ T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4
+
CD8
+
T cells and γδ T cells.
Delocalization of Vg1 mRNA from the Vegetal Cortex in
Xenopus
Oocytes After Destruction of Xlsirt RNA
Authors: Kloc Malgorzata , Etkin Laurence D.
Published: October 2006

The Xlsirts are a family of transcribed repeat sequence genes that do not code for protein. Xlsirt RNAs become localized to the vegetal cortex of
Xenopus
oocytes early in oogenesis, before the localization of the messenger RNA Vg1, which encodes a transforming growth factor-β-like molecule involved in mesoderm formation, and coincident with the localization of Xcat2 transcripts, which encode a nanos-like molecule. Destruction of the localized Xlsirts by injection of antisense oligodeoxynucleotides into stage 4 oocytes resulted in the release of Vg1 transcripts but not Xcat2 transcripts from the vegetal cortex. Xlsirt RNAs, which may be a structural component of the vegetal cortex, are a crucial part of a genetic pathway necessary for the proper localization of Vg1 that leads to subsequent normal pattern formation.
Regulatory T Cell Clones Induced by Oral Tolerance: Suppression of Autoimmune Encephalomyelitis
Authors: Chen Youhai , Kuchroo Vijay K. , Inobe Jun-ichi , Hafler David A. , Weiner Howard L.
Published: October 2006

Experimental autoimmune encephalomyelitis (EAE) is a cell-mediated autoimmune disease that serves as an animal model for multiple sclerosis. Oral administration of myelin basic protein (MBP) suppresses EAE by inducing peripheral tolerance. T cell clones were isolated from the mesenteric lymph nodes of SJL mice that had been orally tolerized to MBP. These clones were CD4
+
and were structurally identical to T helper cell type 1 (T
H
1) encephalitogenic CD4
+
clones in T cell receptor usage, major histocompatibility complex restriction, and epitope recognition. However, they produced transforming growth factor-β with various amounts of interleukin-4 and interleukin-10 and suppressed EAE induced with either MBP or proteolipid protein. Thus, mucosally derived T
H
2-like clones induced by oral antigen can actively regulate immune responses in vivo and may represent a different subset of T cells.
Control of Angiogenesis in Fibroblasts by p53 Regulation of Thrombospondin-1
Authors: Dameron Kristina M. , Volpert Olga V. , Tainsky Michael A. , Bouck Noël
Published: October 2006

As normal cells progress toward malignancy, they must switch to an angiogenic phenotype to attract the nourishing vasculature that they depend on for their growth. In cultured fibroblasts from Li-Fraumeni patients, this switch was found to coincide with loss of the wild-type allele of the p53 tumor suppressor gene and to be the result of reduced expression of thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis. Transfection assays revealed that p53 can stimulate the endogenous TSP-1 gene and positively regulate TSP-1 promoter sequences. These data indicate that, in fibroblasts, wild-type p53 inhibits angiogenesis through regulation of TSP-1 synthesis.
Two Identical Noninteracting Sites in an Ion Channel Revealed by Proton Transfer
Authors: Root Michael J. , MacKinnon Roderick
Published: October 2006

The functional consequences of single proton transfers occurring in the pore of a cyclic nucleotide-gated channel were observed with patch recording techniques. These results led to three conclusions about the chemical nature of ion binding sites in the conduction pathway: The channel contains two identical titratable sites, even though there are more than two (probably four) identical subunits; the sites are formed by glutamate residues that have a p
K
a
(where
K
a
is the acid constant) of 7.6; and protonation of one site does not perturb the p
K
a
of the other. These properties point to an unusual arrangement of carboxyl side-chain residues in the pore of a cation channel.
Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection
Authors: Nilsson Mats , Malmgren Helena , Samiotaki Martina , Kwiatkowski Marek , Chowdhary Bhanu P. , Landegren Ulf
Published: October 2006

Nucleotide sequence information derived from DNA segments of the human and other genomes is accumulating rapidly. However, it frequently proves difficult to use such short DNA segments to identify clones in genomic libraries or fragments in blots of the whole genome or for in situ analysis of chromosomes. Oligonucleotide probes, consisting of two target-complementary segments, connected by a linker sequence, were designed. Upon recognition of the specific nucleic acid molecule the ends of the probes were joined through the action of a ligase, creating circular DNA molecules catenated to the target sequence. These probes thus provide highly specific detection with minimal background.
Alignment and Sensitive Detection of DNA by a Moving Interface
Authors: Bensimon A. , Simon A. , Chiffaudel A. , Croquette V. , Heslot F. , Bensimon D.
Published: October 2006

In a process called "molecular combing," DNA molecules attached at one end to a solid surface were extended and aligned by a receding air-water interface and left to dry on the surface. Molecular combing was observed to extend the length of the bacteriophage λ DNA molecule to 21.5 ± 0.5 micrometers (unextended length, 16.2 micrometers). With the combing process, it was possible to (i) extend a chromosomal
Escherichia coli
DNA fragment (10
6
base pairs) and (ii) detect a minute quantity of DNA (10
3
molecules). These results open the way for a faster physical mapping of the genome and for the detection of small quantities of target DNA from a population of molecules.
Rescue of T Cell-Specific V(D)J Recombination in SCID Mice by DNA-Damaging Agents
Authors: Danska Jayne S. , Pflumio Françoise , Williams Christine J. , Huner Ozgur , Dick John E. , Guidos Cynthia J.
Published: October 2006

Assembly of antigen receptor V (variable), D (diversity), and J (joining) gene segments requires lymphocyte-specific genes and ubiquitous DNA repair activities. Severe combined immunodeficient (SCID) mice are defective in general double-strand (ds) DNA break repair and V(D)J coding joint formation, resulting in arrested lymphocyte development. A single treatment of newborn SCID mice with DNA-damaging agents restored functional, diverse, T cell receptor β chain coding joints, as well as development and expansion of thymocytes expressing both CD4 and CD8 coreceptors, but did not promote B cell development. Thymic lymphoma developed in all mice treated with DNA-damaging agents, suggesting an interrelation between V(D)J recombination, dsDNA break repair, and lymphomagenesis.
A Low-Barrier Hydrogen Bond in the Catalytic Triad of Serine Proteases
Authors: Frey Perry A. , Whitt Sean A. , Tobin John B.
Published: October 2006

Spectroscopic properties of chymotrypsin and model compounds indicate that a low-barrier hydrogen bond participates in the mechanism of serine protease action. A low-barrier hydrogen bond between Nδ1 of His
57
and the β-carboxyl group of Asp
102
in chymotrypsin can facilitate the formation of the tetrahedral adduct, and the nuclear magnetic resonance properties of this proton indicate that it is a low-barrier hydrogen bond. These conclusions are supported by the chemical shift of this proton, the deuterium isotope effect on the chemical shift, and the properties of hydrogen-bonded model compounds in organic solvents, including the hydrogen bond in
cis
-urocanic acid, in which the imidazole ring is internally hydrogen-bonded to the carboxyl group.
Roles of N-Type and Q-Type Ca
2+
Channels in Supporting Hippocampal Synaptic Transmission
Authors: Wheeler David B. , Randall Andrew , Tsien Richard W.
Published: October 2006

Several types of calcium channels found in the central nervous system are possible participants in triggering neurotransmitter release. Synaptic transmission between hippocampal CA3 and CA1 neurons was mediated by N-type calcium channels, together with calcium channels whose pharmacology differs from that of L- and P-type channels but resembles that of the Q-type channel encoded by the α
1A
subunit gene. Blockade of either population of channels strongly increased enhancement of synaptic transmission with repetitive stimuli. Even after complete blockade of N-type channels, transmission was strongly modulated by stimulation of neurotransmitter receptors or protein kinase C. These findings suggest a role for α
1A
subunits in synaptic transmission and support the idea that neurotransmitter release may depend on multiple types of calcium channels under physiological conditions.
Agmatine: an Endogenous Clonidine-Displacing Substance in the Brain
Authors: Li Gen , Regunathan S. , Barrow Colin J. , Eshraghi Jamshid , Cooper Raymond , Reis Donald J.
Published: October 2006

Clonidine, an antihypertensive drug, binds to α
2
-adrenergic and imidazoline receptors. The endogenous ligand for imidazoline receptors may be a clonidine-displacing substance, a small molecule isolated from bovine brain. This clonidine-displacing substance was purified and determined by mass spectroscopy to be agmatine (decarboxylated arginine), heretofore not detected in brain. Agmatine binds to α
2
-adrenergic and imidazoline receptors and stimulates release of catecholamines from adrenal chromaffin cells. Its biosynthetic enzyme, arginine decarboxylase, is present in brain. Agmatine, locally synthesized, is an endogenous agonist at imidazoline receptors, a noncatecholamine ligand at α
2
-adrenergic receptors and may act as a neurotransmitter.
A Subset of CD4
+
Thymocytes Selected by MHC Class I Molecules
Authors: Bendelac Albert , Killeen Nigel , Littman Dan R. , Schwartz Ronald H.
Published: October 2006

To complete their maturation, most immature thymocytes depend on the simultaneous engagement of their antigen receptor [αβ T cell receptor (TCR)] and their CD4 or CD8 coreceptors with major histocompatibility complex class II or I ligands, respectively. However, a normal subset of mature αβ TCR+ thymocytes did not follow these rules. These thymocytes expressed NK1.1 and a restricted set of αβ TCRs that are intrinsically class I-reactive because their positive selection was class I-dependent but CD8-independent. These cells were CD4
+
and CD4
-
8
-
but never CD8
+
, because the presence of CD8 caused negative selection. Thus, neither CD4 nor CD8 contributes signals that direct their maturation into the CD4
+
and CD4
-
8
-
lineages.
Characterization of a Functional GroEL
14
(GroES
7
)
2
Chaperonin Hetero-Oligomer
Authors: Azem Abdussalam , Kessel Martin , Goloubinoff Pierre
Published: October 2006

Chaperonins GroEL and GroES form two types of hetero-oligomers in vitro that can mediate the folding of proteins. Chemical cross-linking and electron microscopy showed that in the presence of adenosine triphosphate (ATP), two GroES
7
rings can successively bind a single GroEL
14
core oligomer. The symmetric GroEL
14
(GroES
7
)
2
chaperonin, whose central cavity appears obstructed by two GroES
7
rings, can nonetheless stably bind and assist the ATP-dependent refolding of RuBisCO enzyme. Thus, unfolded proteins first bind and possibly fold on the external envelope of the chaperonin hetero-oligomer.
Kin Selection, Social Structure, Gene Flow, and the Evolution of Chimpanzees
Authors: Morin Phillip A. , Moore James J. , Chakraborty Ranajit , Jin Li , Goodall Jane , Woodruff David S.
Published: October 2006

Hypotheses about chimpanzee social behavior, phylogeography, and evolution were evaluated by noninvasive genotyping of free-ranging individuals from 20 African sites. Degrees of relatedness among individuals in one community were inferred from allele-sharing at eight nuclear simple sequence repeat (SSR) loci. Males are related on the order of half-siblings, and homozygosity is significantly increased at several SSR loci compared to Hardy-Weinberg expectations. These data support the kin-selection hypothesis for the evolution of cooperation among males. Sequence variation patterns at two mitochondrial loci indicate historically high long-distance gene flow and clarify the relationships among three allopatric subspecies. The unexpectedly large genetic distance between the western subspecies,
Pan troglodytes verus
, and the other two subspecies suggests a divergence time of about 1.58 million years. This result, if confirmed at nuclear loci and supported by eco-behavioral data, implies that
P. t. verus
should be elevated to full species rank.
Regulation of IgE Responses to Inhaled Antigen in Mice by Antigen-Specific γδ T Cells
Authors: McMenamin Christine , Pimm Carolyn , McKersey Michelle , Holt Patrick G.
Published: October 2006

Indirect evidence implicates γδ T cells in the cross-regulation of CD4 αβ T cell responses. Adoptive transfer of small numbers of γδ T cells from ovalbumin (OVA)-tolerant mice selectively suppressed T
H
2 -dependent immunoglobulin E(IgE) antibody production without affecting parallel IgG responses. Challenge of these γδ T cells in vitro with specific antigen resulted in production of high levels of interferon γ. The effects of the γδ T cells may be mediated by direct inhibition of OVA-specific CD4
+
T
H
2 cell proliferation or selection for specific CD4 T
H
2 cells.
Mediation of Hippocampal Mossy Fiber Long-Term Potentiation by Cyclic AMP
Authors: Weisskopf Marc G. , Castillo Pablo E. , Zalutsky Robert A. , Nicoll Roger A.
Published: October 2006

Repetitive activation of hippocampal mossy fibers evokes a long-term potentiation (LTP) of synaptic responses in pyramidal cells in the CA3 region that is independent of
N
-methyl-D-aspartate receptor activation. Previous results suggest that the site for both the induction and expression of this form of LTP is presynaptic. Experimental elevation of cyclic adenosine 3′,5′-monophosphate (cAMP) both mimics and interferes with tetanus-induced mossy fiber LTP, and blockers of the cAMP cascade block mossy fiber LTP. It is proposed that calcium entry into the presynaptic terminal may activate Ca
2+
-calmodulin-sensitive adenylyl cyclase I which, through protein kinase A, causes a persistent enhancement of evoked glutamate release.
An Experimental Study of Inbreeding Depression in a Natural Habitat
Authors: Jiménez Julie A. , Hughes Kimberly A. , Alaks Glen , Graham Laurie , Lacy Robert C.
Published: October 2006

Inbreeding is known to lead to decreased survival and reproduction in captive populations of animals. It is also important to know whether inbreeding has deleterious effects in natural habitats. An estimate was made of the effects of inbreeding in white-footed mice,
Peromyscus leucopus noveboracensis
, derived from a wild population. This study demonstrates that inbreeding had a significant detrimental effect on the survivorship of mice reintroduced into a natural habitat. This effect was more severe than the effect observed in laboratory studies of the population.
Inhibition of Ras-Induced DNA Synthesis by Expression of the Phosphatase MKP-1
Authors: Sun Hong , Tonks Nicholas K. , Bar-Sagi Dafna
Published: October 2006

Mitogen-activated protein kinases (MAP kinases) are common components of signaling pathways induced by diverse growth stimuli. Although the guanidine nucleotide-binding Ras proteins are known to be upstream activators of MAP kinases, the extent to which MAP kinases directly contribute to the mitogenic effect of Ras is as yet undefined. In this study, inhibition of MAP kinases by the MAP kinase phosphatase MKP-1 blocked the induction of DNA synthesis in quiescent rat embryonic fibroblast REF-52 cells by an activated mutant of Ras, V
12
Ras. These results suggest an essential role for activation of MAP kinases in the transition from the quiescent to the DNA replication phase of the eukaryotic cell cycle.
Vibrationally Coherent Photochemistry in the Femtosecond Primary Event of Vision
Authors: Wang Qing , Schoenlein Robert W. , Peteanu Linda A. , Mathies Richard A. , Shank Charles V.
Published: October 2006

Femtosecond pump-probe experiments reveal the impulsive production of photoproduct in the primary event in vision. The retinal chromophore of rhodopsin was excited with a 35-femtosecond pulse at 500 nanometers, and transient changes in absorption were measured with 10-femtosecond probe pulses. At probe wavelengths within the photoproduct absorption band, oscillatory features with a period of 550 femtoseconds (60 wavenumbers) were observed whose phase and amplitude demonstrate that they are the result of nonstationary vibrational motion in the ground state of the photoproduct. The observation of coherent vibrational motion of the photoproduct supports the idea that the primary step in vision is a vibrationally coherent process and that the high quantum yield of the cis→trans isomerization in rhodopsin is a consequence of the extreme speed of the excited-state torsional motion.
The Structure of Flavocytochrome c Sulfide Dehydrogenase from a Purple Phototrophic Bacterium
Authors: Chen Zhi-wei , Koh Monjoo , Van Driessche Gonzalez , Van Beeumen Jozef J. , Bartsch Robert G. , Meyer Terry E. , Cusanovich Michael A. , Mathews F. Scott
Published: October 2006

The structure of the heterodimeric flavocytochrome c sulfide dehydrogenase from
Chromatium vinosum
was determined at a resolution of 2.53 angstroms. It contains a glutathione reductase-like flavin-binding subunit and a diheme cytochrome subunit. The diheme cytochrome folds as two domains, each resembling mitochondrial cytochrome c, and has an unusual interpropionic acid linkage joining the two heme groups in the interior of the subunit. The active site of the flavoprotein subunit contains a catalytically important disulfide bridge located above the pyrimidine portion of the flavin ring. A tryptophan, threonine, or tyrosine side chain may provide a partial conduit for electron transfer to one of the heme groups located 10 angstroms from the flavin.
Epidermal Cell Fate Determination in
Arabidopsis
: Patterns Defined by a Steroid-Inducible Regulator
Authors: Lloyd Alan M. , Schena Mark , Walbot Virginia , Davis Ronald W.
Published: October 2006

The
Arabidopsis
mutant
ttg
lacks both trichomes (epidermal hairs) and anthocyanin pigments. Trichomes and anthocyanins are restored by the constitutive expression of the maize transcriptional regulator (R). The expression of an R-glucocorticoid receptor chimeric protein results in a steroid hormone-dependent, conditional allele of R that functions in whole
Arabidopsis
plants. The response of the chimeric protein to pulses of hormone was used to define the pattern and timing of trichome formation on the developing leaf epidermis. Each adaxial epidermal leaf cell appears to have an equal probability of differentiating into a trichome; there is a temporal zone of decision for trichome cell fate that proceeds as a wave from the tip to the base of developing leaves.
Vancomycin Resistance: Structure of D-Alanine:D-Alanine Ligase at 2.3 Å Resolution
Authors: Fan Chang , Moews Paul C. , Walsh Christopher T. , Knox James R.
Published: October 2006

The molecular structure of the D-alanine:D-alanine ligase of the
ddlB
gene of
Escherichia coli
, co-crystallized with an
S
,
R
-methylphosphinate and adenosine triphosphate, was determined by x-ray diffraction to a resolution of 2.3 angstroms. A catalytic mechanism for the ligation of two D-alanine substrates is proposed in which a helix dipole and a hydrogen-bonded triad of tyrosine, serine, and glutamic acid assist binding and deprotonation steps. From sequence comparison, it is proposed that a different triad exists in a recently discovered D-alanine:D-lactate ligase (VanA) present in vancomycin-resistant enterococci. A molecular mechanism for the altered specificity of VanA is suggested.
c-Fos: a Key Regulator of Osteoclast-Macrophage Lineage Determination and Bone Remodeling
Authors: Grigoriadis Agamemnon E. , Wang Zhao-Qi , Cecchini Marco G. , Hofstetter Willy , Felix Rolf , Fleisch Herbert A. , Wagner Erwin F.
Published: October 2006

Mice lacking the proto-oncogene c-fosdevelop the bone disease osteopetrosis. Fos mutant mice were found to have a block in the differentiation of bone-resorbing osteoclasts that was intrinsic to hematopoietic cells. Bone marrow transplantation rescued the osteopetrosis, and ectopic c-fosexpression overcame this differentiation block. The lack of Fos also caused a lineage shift between osteoclasts and macrophages that resulted in increased numbers of bone marrow macrophages. These results identify Fos as a key regulator of osteoclast-macrophage lineage determination in vivo and provide insights into the molecular mechanisms underlying metabolic bone diseases.
Developmental Evolution: Insights from Studies of Insect Segmentation
Authors: Patel Nipam H.
Published: October 2006

Rapid advances have been made in the understanding of the genetic basis of development and pattern formation in a variety of model systems. By examining the extent to which these developmental systems are conserved or altered between different organisms, insight can be gained into the evolutionary events that have generated the diversity of organisms around us. The molecular and genetic basis of early pattern formation in
Drosophila melanogaster
has been particularly well studied, and comparisons to other insects have revealed conservation of some aspects of development, as well as differences that may explain variations in early patterning events.
Cytoskeletal Functions During
Drosophila
Oogenesis
Authors: Cooley Lynn , Theurkauf William E.
Published: October 2006

Organismal morphogenesis is driven by a complex series of developmentally coordinated changes in cell shape, size, and number. These changes in cell morphology are in turn dependent on alterations in basic cytoarchitecture. Elucidating the mechanisms of development thus requires an understanding of the cytoskeletal elements that organize the cytoplasm of differentiating cells.
Drosophila
oogenesis has emerged as a versatile system for the study of cytoskeletal function during development. A series of highly coordinated changes in cytoskeletal organization are required to produce a mature
Drosophila
oocyte, and these cytoskeletal transformations are amenable to a variety of experimental approaches. Genetic, molecular, and cytological studies have shed light on the specific functions of the cytoskeleton during oogenesis. The results of these studies are reviewed here, and their mechanistic implications are considered.
Observation of Coherent Reaction Dynamics in Heme Proteins
Authors: Zhu Leyun , Sage J. Timothy , Champion Paul M.
Published: October 2006

Femtosecond laser pulses, resonant with the Soret band of the nitric oxide complex of myoglobin (MbNO), were used to probe coherent, low-frequency nuclear motion of the heme group after photolysis. Distinct oscillations with periods of 430 and 150 femtoseconds were observed and are attributed to heme doming and iron-histidine motion, respectively. These results verify that the nuclear motion of the heme is strongly coupled to the ligand binding reaction and demonstrate that such motion is not determined by overdamped (diffusive) dynamics. The relative phases and frequencies of the nuclear motion of the photoproduct suggest that the coherence arises from impulsive electronic forces associated with the spin-state change of the heme iron atom and the depopulation of its dz2 orbital during the bond-breaking event.
An AIDS-Like Condition Induced in Baboons by HIV-2
Authors: Barnett Susan W. , Murthy Krishna K. , Herndier Brian G. , Levy Jay A.
Published: October 2006

Six baboons (
Papio cynocephalus
) were intravenously inoculated with the human immunodeficiency virus-type 2 (HIV-2) strain HIV-2
UC2
. All seroconverted within 6 weeks after inoculation; five animals became persistently infected. Four developed lymphadenopathy, and three of the animals had CD4
+
T cell loss within 18 to 24 months after inoculation. One of these baboons, showing severe clinical symptoms, showed at necropsy widespread dissemination of virus with follicular depletion in the lymph nodes, extensive fibromatosis involving lymphoid and nonlymphoid tissues, and lymphocytic interstitial pneumonitis. Another animal is cachectic and exhibited lymphoid follicular lysis and fibrous skin lesions. Other baboons inoculated with a second strain, HIV-2
UC14
, have shown evidence of persistent infection. HIV-2 infection of baboons provides a valuable animal model for studying HIV persistence and pathogenesis and for evaluating approaches to antiviral therapies.
Crystal Structure of LacI Member, PurR, Bound to DNA: Minor Groove Binding by α Helices
Authors: Schumacher Maria A. , Choi Kang Yell , Zalkin Howard , Brennan Richard G.
Published: October 2006

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair
purF
operator site has been solved at 2.7 Å resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related α helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu
54
and its symmetry-related mate, Leu
54′
, into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the
purF
operator by 45 degrees.
Synthesis of Proteins by Native Chemical Ligation
Authors: Dawson Philip E. , Muir Tom W. , Clark-Lewis Ian , Kent Stephen B. H.
Published: October 2006

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.
A Three-Dimensional Model for the Hammerhead Ribozyme Based on Fluorescence Measurements
Authors: Tuschl Thomas , Gohlke Christoph , Jovin Thomas M. , Westhof Eric , Eckstein Fritz
Published: October 2006

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.
Hydroxyurea as an Inhibitor of Human Immunodeficiency Virus-Type 1 Replication
Authors: Lori Franco , Malykh Andrei , Cara Andrea , Sun Daisy , Weinstein John N. , Lisziewicz Julianna , Gallo Robert C.
Published: October 2006

Hydroxyurea, a drug widely used in therapy of several human diseases, inhibits deoxynucleotide synthesis—and, consequently, DNA synthesis—by blocking the cellular enzyme ribonucleotide reductase. Hydroxyurea inhibits human immunodeficiency virus-type 1 (HIV-1) DNA synthesis in activated peripheral blood lymphocytes by decreasing the amount of intracellular deoxynucleotides, thus suggesting that this drug has an antiviral effect. Hydroxyurea has now been shown to block HIV-1 replication in acutely infected primary human lymphocytes (quiescent and activated) and macrophages, as well as in blood cells infected in vivo obtained from individuals with acquired immunodeficiency syndrome (AIDS). The antiviral effect was achieved at nontoxic doses of hydroxyurea, lower than those currently used in human therapy. Combination of hydroxyurea with the nucleoside analog didanosine (2′,3′-dideoxyinosine, or ddl) generated a synergistic inhibitory effect without increasing toxicity. In some instances, inhibition of HIV-1 by hydroxyurea was irreversible, even several weeks after suspension of drug treatment. The indirect inhibition of HIV-1 by hydroxyurea is not expected to generate high rates of escape mutants. Hydroxyurea therefore appears to be a possible candidate for AIDS therapy.
DNA Loop Repair by Human Cell Extracts
Authors: Umar Asad , Boyer Jayne C. , Kunkel Thomas A.
Published: October 2006

An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5′ or 3′ to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type
hMLH1
gene and is deficient in repair of mismatches. However, a cell line with deletions in both
hMSH2
alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.
Molecular Computation of Solutions to Combinatorial Problems
Authors: Adleman Leonard M.
Published: October 2006

The tools of molecular biology were used to solve an instance of the directed Hamiltonian path problem. A small graph was encoded in molecules of DNA, and the "operations" of the computation were performed with standard protocols and enzymes. This experiment demonstrates the feasibility of carrying out computations at the molecular level.
Interaction of IL-2Rβ and γ
c
Chains with Jak1 and Jak3: Implications for XSCID and XCID
Authors: Russell Sarah M. , Johnston James A. , Noguchi Masayuki , Kawamura Masaru , Bacon Chris M. , Friedmann Michael , Berg Maria , McVicar Daniel W. , Witthuhn Bruce A. , Silvennoinen Olli , Goldman Armond S. , Schmalstieg Frank C. , Ihle James N. , O'Shea John J. , Leonard Warren J.
Published: October 2006

Interleukin-2 (IL-2) signaling requires the dimerization of the IL-2 receptor β (IL-2Rβ) and common γ (γ
c
) chains. Mutations of γ
c
can result in X-linked severe combined immunodeficiency (XSCID). IL-2, IL-4, IL-7 (whose receptors are known to contain γ
c
), and IL-9 (whose receptor is shown here to contain γ
c
) induced the tyrosine phosphorylation and activation of the Janus family tyrosine kinases Jak1 and Jak3. Jak1 and Jak3 associated with IL-2Rβ and γ
c
, respectively; IL-2 induced Jak3-IL-2Rβ and increased Jak3-γ
c
associations. Truncations of γ
c
, and a γ
c
, point mutation causing moderate X-linked combined immunodeficiency (XCID), decreased γ
c
-Jak3 association. Thus, γ
c
mutations in at least some XSCID and XCID patients prevent normal Jak3 activation, suggesting that mutations of Jak3 may result in an XSCID-like phenotype.
Spermine and Spermidine as Gating Molecules for Inward Rectifier K
+
Channels
Authors: Ficker Eckhard , Taglialatela Maurizio , Wible Barbara A. , Henley Charles M. , Brown Arthur M.
Published: October 2006

Inward rectifier K
+
channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg
2+
and a Mg
2+
-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K
+
channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.
A Phase of Liposomes with Entangled Tubular Vesicles
Authors: Chiruvolu Shivkumar , Warriner Heidi E. , Naranjo Edward , Idziak Stefan H. J. , Rädler Joachim , Plano Robert J. , Zasadzinski Joseph A. , Safinya Cyrus R.
Published: October 2006

An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled L
tv
, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the L
tv
phase coexists with the well-known L
4
phase of spherical vesicles and a bulk L
α
phase. However, the defining characteristic of the L
tv
phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure.
Circadian Clock Mutants of Cyanobacteria
Authors: Kondo Takao , Tsinoremas Nicholas F. , Golden Susan S. , Johnson Carl Hirschie , Kutsuna Shinsuke , Ishiura Masahiro
Published: October 2006

A diverse set of circadian clock mutants was isolated in a cyanobacterial strain that carries a bacterial luciferase reporter gene attached to a clock-controlled promoter. Among 150,000 clones of chemically mutagenized bioluminescent cells, 12 mutants were isolated that exhibit a broad spectrum of periods (between 16 and 60 hours), and 5 mutants were found that show a variety of unusual patterns, including arrhythmia. These mutations appear to be clock-specific. Moreover, it was demonstrated that in this cyanobacterium it is possible to clone mutant genes by complementation, which provides a means to genetically dissect the circadian mechanism.
Activation and Regeneration of Rhodopsin in the Insect Visual Cycle
Authors: Kiselev Alexander , Subramaniam Sriram
Published: October 2006

Light absorption by rhodopsin generates metarhodopsin, which activates heterotrimeric guanine nucleotide-binding proteins (G proteins) in photoreceptor cells of vertebrates and invertebrates. In contrast to vertebrate metarhodopsins, most invertebrate metarhodopsins are thermally stable and regenerate rhodopsin by absorption of a second photon. In experiments with Rh1Drosophilarhodopsin, the thermal stability of metarhodopsin was found not to be an intrinsic property of the visual pigment but a consequence of its interaction with arrestin (49 kilodaltons). The stabilization of metarhodopsin resulted in a large decrease in the efficiency of G protein activation. Light absorption by thermally stable metarhodopsin initially regenerated an inactive rhodopsin-like intermediate, which was subsequently converted in the dark to active rhodopsin. The accumulation of inactive rhodopsin at higher light levels may represent a mechanism for gain regulation in the insect visual cycle.
Cell Cycle Control by a Complex of the Cyclin HCS26 (PCL1) and the Kinase PHO85
Authors: Espinoza F. Hernan , Ogas Joseph , Herskowitz Ira , Morgan David O.
Published: October 2006

The events of the eukaryotic cell cycle are governed by cyclin-dependent kinases (cdk's), whose activation requires association with cyclin regulatory subunits expressed at specific cell cycle stages. In the budding yeast
Saccharomyces cerevisiae
, the cell cycle is thought to be controlled by a single cdk, CDC28. Passage through the G
1
phase of the cell cycle is regulated by complexes of CDC28 and G
1
cyclins (CLN1, CLN2, and CLN3). A putative G
1
cyclin, HCS26, has recently been identified. In a/α diploid cells lacking CLN1 and CLN2, HCS26 is required for passage through G
1
. HCS26 does not associate with CDC28, but instead associates with PHO85, a closely related protein kinase. Thus, budding yeast, like higher eukaryotes, use multiple cdk's in the regulation of cell cycle progression.
Binding of Mismatched Microsatellite DNA Sequences by the Human MSH2 Protein
Authors: Fishel Richard , Ewel Amy , Lee Suman , Lescoe Mary Kay , Griffith Jack
Published: October 2006

Alteration of the human mismatch repair gene
hMSH2
has been linked to the microsatellite DNA instability found in hereditary nonpolyposis colon cancer and several sporadic cancers. This microsatellite DNA instability is thought to arise from defective repair of DNA replication errors that create insertion-deletion loop-type (IDL) mismatched nucleotides. Here, it is shown that purified hMSH2 protein efficiently and specifically binds DNA containing IDL mismatches of up to 14 nucleotides. These results support a direct role for hMSH2 in mutation avoidance and microsatellite stability in human cells.
Differential Activation of ERK and JNK Mitogen-Activated Protein Kinases by Raf-1 and MEKK
Authors: Minden Audrey , Lin Anning , McMahon Martin , Lange-Carter Carol , Dérijard Benoit , Davis Roger J. , Johnson Gary L. , Karin Michael
Published: October 2006

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor α (TNF-α) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades—one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.
Cell Cycle Control and Cancer
Authors: Hartwell Leland H. , Kastan Michael B.
Published: October 2006

Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.
DNA Bending by Asymmetric Phosphate Neutralization
Authors: Strauss Juliane K. , Maher L. James
Published: October 2006

DNA is often bent when complexed with proteins. Understanding the forces responsible for DNA bending would be of fundamental value in exploring the interplay of these macromolecules. A series of experiments was devised to test the hypothesis that proteins with cationic surfaces can induce substantial DNA bending by neutralizing phosphates on one DNA face. Repulsions between phosphates in the remaining anionic helix are predicted to result in an unbalanced compression force acting to deform the DNA toward the protein. This hypothesis is supported by the results of electrophoretic experiments in which DNA spontaneously bends when one helical face is partially modified by incorporation of neutral phosphate analogs. Phasing with respect to a site of intrinsic DNA curvature (hexadeoxyadenylate tract) permits estimation of the electrostatic bend angle, and demonstrates that such modified DNAs are deformed toward the neutralized surface, as predicted. Similar model systems may be useful in exploring the extent to which phosphate neutralization can account for DNA bending by particular proteins.
Toxic Shock Syndrome Toxin-1 Complexed with a Class II Major Histocompatibility Molecule HLA-DR1
Authors: Kim Jongsun , Urban Robert G. , Strominger Jack L. , Wiley Don C.
Published: October 2006

The three-dimensional structure of a
Staphylococcus aureus
superantigen, toxic shock syndrome toxin-1 (TSST-1), complexed with a human class II major histocompatibility molecule (DR1), was determined by x-ray crystallography. The TSST-1 binding site on DR1 overlaps that of the superantigen
S. aureus
enterotoxin B (SEB), but the two binding modes differ. Whereas SEB binds primarily off one edge of the peptide binding site of DR1, TSST-1 extends over almost one-half of the binding site and contacts both the flanking α helices of the histocompatibility antigen and the bound peptide. This difference suggests that the T cell receptor (TCR) would bind to TSST-1:DR1 very differently than to DR1:peptide or SEB:DR1. It also suggests that TSST-1 binding may be dependent on the peptide, though less so than TCR binding, providing a possible explanation for the inability of TSST-1 to competitively block SEB binding to all DR1 molecules on cells (even though the binding sites of TSST-1 and SEB on DR1 overlap almost completely) and suggesting the possibility that T cell activation by superantigen could be directed by peptide antigen.
Mathematical Studies of Parasitic Infection and Immunity
Authors: Anderson Roy M.
Published: October 2006

The techniques that underpin modern molecular biology have been rapidly adopted by those interested in the major parasitic infections of humans. The parasitological literature is full of reports of genes and their amino acid sequences, of molecules, of cell membrane receptors and channels, and of the fine details of the immunological responses mounted by the host to combat infection. Much less enthusiasm has been shown for the mathematical techniques that facilitate the analysis and interpretation of dynamical processes such as transmission, evolution, and the interplay between parasite population growth and immunological responses within the host. Molecular techniques provide enormous opportunities for description, but ultimately, understanding biological systems with the precision that physicists and engineers aspire to in their own fields will require quantitative description of the many rate processes that dictate both an observed pattern and the dynamics of its change.
Low-Barrier Hydrogen Bonds and Enzymic Catalysis
Authors: Cleland W. W. , Kreevoy Maurice M.
Published: October 2006

Formation of a short (less than 2.5 angstroms), very strong, low-barrier hydrogen bond in the transition state, or in an enzyme-intermediate complex, can be an important contribution to enzymic catalysis. Formation of such a bond can supply 10 to 20 kilocalories per mole and thus facilitate difficult reactions such as enolization of carboxylate groups. Because low-barrier hydrogen bonds form only when the p
K
a
's (negative logarithm of the acid constant) of the oxygens or nitrogens sharing the hydrogen are similar, a weak hydrogen bond in the enzyme-substrate complex in which the p
K
a
's do not match can become a strong, low-barrier one if the p
K
a
's become matched in the transition state or enzyme-intermediate complex. Several examples of enzymatic reactions that appear to use this principle are presented.
Functional Role of Type I and Type II Interferons in Antiviral Defense
Authors: Müller Ulrike , Steinhoff Ulrich , Reis Luiz F. L. , Hemmi Silvio , Pavlovic Jovan , Zinkernagel Rolf M. , Aguet Michel
Published: October 2006

Mice lacking the known subunit of the type I interferon (IFN) receptor were completely unresponsive to type I IFNs, suggesting that this receptor chain is essential for type I IFN-mediated signal transduction. These mice showed no overt anomalies but were unable to cope with viral infections, despite otherwise normal immune responses. Comparison of mice lacking either type I or type II IFN receptors showed that, at least in response to some viruses, both IFN systems are essential for antiviral defense and are functionally nonredundant.
Involvement of the IRF-1 Transcription Factor in Antiviral Responses to Interferons
Authors: Kimura Tohru , Nakayama Katsutoshi , Penninger Josef , Kitagawa Motoo , Harada Hisashi , Matsuyama Toshifumi , Tanaka Nobuyuki , Kamijo Ryutaro , Vilček Jan , Mak Tak W. , Taniguchi Tadatsugu
Published: October 2006

The mechanisms underlying interferon (IFN)-induced antiviral states are not well understood. Interferon regulatory factor-1 (IRF-1) is an IFN-inducible transcriptional activator, whereas IRF-2 suppresses IRF-1 action. The inhibition of encephalomyocarditis virus (EMCV) replication by IFN-α and especially by IFN-γ was impaired in cells from mice with a null mutation in the
IRF-1
gene (IRF-1
-/-
mice). The IRF-1
-/-
mice were less resistant than normal mice to EMCV infection, as revealed by accelerated mortality and a larger virus titer in target organs. The absence of IRF-1 did not clearly affect replication of two other types of viruses. Thus, IRF-1 is necessary for the antiviral action of IFNs against some viruses, but IFNs activate multiple activation pathways through diverse target genes to induce the antiviral state.