bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting ligand-binding function in families of bacterial receptors
Authors: Johnson Jason M. , Church George M.
Published: July 2002

The three-dimensional fold of a new protein sequence can often be inferred directly from sequence homology to a protein of known structure. The function of a new protein sequence is more difficult to predict, however, since homologues can have different molecular and cellular functions. To develop and automate computational methods for determining molecular function, we have analyzed ligand-binding specificity in two related families of binding proteins. One of these families includes
Escherichia coli
lactose repressor and ribose-binding protein, and the other includes
E. coli
sulfate- and phosphate-binding proteins. These proteins have similar folds but varying specificity, binding many different small molecules, including mono- and disaccharides, purines, oxyanions, ferric iron, and polyamines. Starting from template structural alignments, alignments of over 90 sequences per family were generated by iterative database searches with hidden Markov models. Phylogenetic trees were made of full-length sequences and of subsets of residues lining the binding cleft, to determine whether subbranches of the trees correlate with ligand-binding preference. Automated analyses of residues in the binding pocket were also used to predict ligand-binding function for many uncharacterized database sequences and to identify specific side chain–ligand contacts in proteins without solved structures. Our results demonstrate the utility of anchoring functional annotation within a protein family context.
Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O
Authors: Walev Iwan , Bhakdi Sebastian Chakrit , Hofmann Fred , Djonder Nabil , Valeva Angela , Aktories Klaus , Bhakdi Sucharit
Published: July 2002

The pore-forming toxin streptolysin O (SLO) can be used to
reversibly permeabilize adherent and nonadherent cells, allowing
delivery of molecules with up to 100 kDa mass to the cytosol. Using
FITC-labeled albumin, 10
5
–10
6
molecules were
estimated to be entrapped per cell. Repair of toxin lesions depended on
Ca
2+
-calmodulin and on intact microtubules, but
was not sensitive to actin disruption or to inhibition of protein
synthesis. Resealed cells were viable for days and retained the
capacity to endocytose and to proliferate. The active domains of large
clostridial toxins were introduced into three different cell lines. The
domains were derived from
Clostridium difficile
B-toxin
and
Clostridium sordelli
lethal toxin, which glycosylate
small G-proteins, and from
Clostridium botulinum
C2
toxin, which ADP-ribosylates actin. After delivery with SLO, all three
toxins disrupted the actin cytoskeleton to cause rounding up of the
cells. Glucosylation assays demonstrated that G-proteins Rho and Ras
were retained in the permeabilized cells and were modified by the
respective toxins. Inactivation of these G-proteins resulted in reduced
stimulus-dependent granule secretion, whereas ADP-ribosylation of actin
by the
C. botulinum
C2-toxin resulted in enhanced
secretion in cells. The presented method for introducing proteins into
living cells should find multifaceted application in cell biology.
Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast
Authors: Boldogh Istvan R. , Yang Hyeong-Cheol , Nowakowski W. Dan , Karmon Sharon L. , Hays Lara G. , Yates John R. , Pon Liza A.
Published: July 2002

The Arp2/3 complex is implicated in actin
polymerization-driven movement of
Listeria
monocytogenes
. Here, we find that Arp2p and Arc15p, two
subunits of this complex, show tight, actin-independent association
with isolated yeast mitochondria. Arp2p colocalizes with mitochondria.
Consistent with this result, we detect Arp2p-dependent formation of
actin clouds around mitochondria in intact yeast. Cells bearing
mutations in
ARP2
or
ARC15
genes show
decreased velocities of mitochondrial movement, loss of all directed
movement and defects in mitochondrial morphology. Finally, we observe a
decrease in the velocity and extent of mitochondrial movement in yeast
in which actin dynamics are reduced but actin cytoskeletal structure is
intact. These results support the idea that the movement of
mitochondria in yeast is actin polymerization driven and that this
movement requires Arp2/3 complex.
Cloning of nitroalkane oxidase from
Fusarium oxysporum
identifies a new member of the acyl-CoA dehydrogenase superfamily
Authors: Daubner S. Colette , Gadda Giovanni , Valley Michael P. , Fitzpatrick Paul F.
Published: July 2002

The flavoprotein nitroalkane oxidase (NAO) from
Fusarium
oxysporum
catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an
F. oxysporum
genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an
F. oxysporum
cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in
Escherichia coli
and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from
F. oxysporum
but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from
E. coli
or from
F. oxysporum
has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.
Impairment of synaptic transmission by transient hypoxia in hippocampal slices: Improved recovery in glutathione peroxidase transgenic mice
Authors: Furling Denis , Ghribi Othman , Lahsaini Ahmed , Mirault Marc-Edouard , Massicotte Guy
Published: July 2002

There is increasing evidence that oxygen free radicals contribute to ischemic brain injury. It is unclear, however, to what extent specific antioxidant enzymes can prevent or reverse the impairment of synaptic function caused by transient hypoxia. In this study, we investigated in transgenic (Tg) mice whether a moderate increase in glutathione peroxidase-1 (GPx1) may improve the capacity of CA1 pyramidal cells to recover synaptic transmission after a short period of hypoxia
in vitro
. In control hippocampal slices, transient hypoxia (7–9 min) produced irreversible loss of excitatory postsynaptic potentials. Complete recovery of synaptic transmission was observed with homozygous Tg-MT-GPx-6 mice after reoxygenation, and, after repeated episodes of hypoxia, synaptic transmission was still viable in most Tg slices, in contrast to non-Tg slices. Moreover, hypoxic episodes abolished the capacity of hippocampal slices to generate long-term potentiation in area CA1 of control mice, whereas a significant extent of long-term potentiation expression was still preserved in Tg tissues. We also demonstrated that susceptibility to
N-
methyl-
d
-aspartate-mediated oxidative injury was reduced in Tg hippocampal slices. In conclusion, our results suggest that a moderate GPx increase can be sufficient to prevent irreversible functional damage produced by transient hypoxia in the hippocampus and to help maintain basic electrophysiological mechanisms involved in memory formation.
The quantity of nitric oxide released by macrophages regulatesChlamydia-induced disease
Authors: Huang Jin , DeGraves Fred J. , Lenz Stephen D. , Gao Dongya , Feng Pu , Li Dan , Schlapp Tobias , Kaltenboeck Bernhard
Published: July 2002

Intracellular bacteria of the genusChlamydiacause numerous typically chronic diseases, frequently with debilitating sequelae. Genetic determinants of disease susceptibility after infection withChlamydiabacteria are unknown. C57BL/6 mice develop severe pneumonia and poor immunity againstChlamydiaafter moderate respiratory infection whereas BALB/c mice are protected from disease and develop vigorous Th1 immunity. Here we show that infected C57BL/6 macrophages release more NO synthesized by NO synthase 2 (NOS2) than BALB/c macrophages and have lower mRNA concentrations of arginase II, a competitor of NOS2 for the common substrate,l-arginine. Reduction, but not elimination, of NO production by incomplete inhibition of NOS2 abolishes susceptibility of C57BL/6 mice toChlamydia-induced disease. Thus, the quantity of NO released by infected macrophages is the effector mechanism that regulates between pathogenic and protective responses to chlamydial infection, and genes controlling NO production determine susceptibility to chlamydial disease.
Suppression of genome instability by redundant S-phase checkpoint pathways in
Saccharomyces
cerevisiae
Authors: Myung Kyungjae , Kolodner Richard D.
Published: July 2002

Cancer cells show increased genome rearrangements, although it is unclear what defects cause these rearrangements. Previous studies have implicated the
Saccharomyces cerevisiae
replication checkpoint in the suppression of spontaneous genome rearrangements. In the present study, low doses of methyl methane sulfonate that activate the intra-S checkpoint but not the G
1
or G
2
DNA damage checkpoints were found to cause increased accumulation of genome rearrangements in both wild-type strains and to an even greater extent in strains containing mutations causing defects in the intra-S checkpoint. The rearrangements were primarily translocations or events resulting in deletion of a portion of a chromosome arm along with the addition of a new telomere. Combinations of mutations causing individual defects in the RAD24 or SGS1 branches of the intra-S checkpoint or the replication checkpoint showed synergistic interactions with regard to the spontaneous genome instability rate. PDS1 and the RAD50–MRE11–XRS2 complex were found to be important members of all the S-phase checkpoints in suppressing genome instability, whereas RAD53 only seemed to play a role in the intra-S checkpoints. Combinations of mutations that seem to result in inactivation of the S-phase checkpoints and critical effectors resulted in as much as 12,000–14,000-fold increases in the genome instability rate. These data support the view that spontaneous genome rearrangements result from DNA replication errors and indicate that there is a high degree of redundancy among the checkpoints that act in S phase to suppress such genome instability.
Pectate lyase A, an enzymatic subunit of the
Clostridium cellulovorans
cellulosome
Authors: Tamaru Yutaka , Doi Roy H.
Published: July 2002

Clostridium cellulovorans
uses not only cellulose
but also xylan, mannan, pectin, and several other carbon sources for
its growth and produces an extracellular multienzyme complex called the
cellulosome, which is involved in plant cell wall degradation. Here we
report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying
downstream of the
engY
gene, which codes for
cellulosomal enzyme EngY.
pelA
is composed of an ORF of
2,742 bp and encodes a protein of 914 aa with a molecular weight of
94,458. The amino acid sequence derived from
pelA
revealed a multidomain structure, i.e., an N-terminal domain partially
homologous to the C terminus of PelB of
Erwinia
chrysanthemi
belonging to family 1 of pectate lyases, a
putative cellulose-binding domain, a catalytic domain homologous to
PelL and PelX of
E. chrysanthemi
that belongs to family
4 of pectate lyases, and a duplicated sequence (or dockerin) at the C
terminus that is highly conserved in enzymatic subunits of the
C. cellulovorans
cellulosome. The recombinant truncated
enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and
trigalacturonic acid (G3) but did not act on G2 and G3. There have been
no reports available to date on pectate lyase genes from
Clostridia
.
Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry
Authors: Miyashita Masahiro , Presley Jack M. , Buchholz Bruce A. , Lam Kit S. , Lee Young Moo , Vogel John S. , Hammock Bruce D.
Published: July 2002

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathioneS-transferase at the attomole level with zeptomole precision using a tracer of14C. The transgenic transferase was labeled by growing transformedEscherichia colion [14C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.
Hydrophobic moments of protein structures: Spatially profiling the distribution
Authors: Silverman B. David
Published: July 2002

It is generally accepted that globular proteins fold with a
hydrophobic core and a hydrophilic exterior. Might the spatial
distribution of amino acid hydrophobicity exhibit common features? The
hydrophobic profile detailing this distribution from the protein
interior to exterior has been examined for 30 relatively diverse
structures obtained from the Protein Data Bank, for 3 proteins of the
30S ribosomal subunit, and for a simple set of 14 decoys. A
second-order hydrophobic moment has provided a simple measure of the
spatial variation. Shapes of the calculated spatial profiles of all
native structures have been found to be comparable. Consequently,
profile shapes as well as particular profile features should assist in
validating predicted protein structures and in discriminating between
different protein-folding pathways. The spatial profiles of the 14
decoys are clearly distinguished from the profiles of their native
structures.
Beyond molecules: Self-assembly of mesoscopic and macroscopic components
Authors: Whitesides George M. , Boncheva Mila
Published: July 2002

Self-assembly is a process in which components, either separate or linked, spontaneously form ordered aggregates. Self-assembly can occur with components having sizes from the molecular to the macroscopic, provided that appropriate conditions are met. Although much of the work in self-assembly has focused on molecular components, many of the most interesting applications of self-assembling processes can be found at larger sizes (nanometers to micrometers). These larger systems also offer a level of control over the characteristics of the components and over the interactions among them that makes fundamental investigations especially tractable.
Xenopus
Kielin: A dorsalizing factor containing multiple chordin-type repeats secreted from the embryonic midline
Authors: Matsui Masaru , Mizuseki Kenji , Nakatani Jin , Nakanishi Shigetada , Sasai Yoshiki
Published: July 2002

The midline tissues are important inductive centers of early vertebrate embryos. By signal peptide selection screening, we isolated a secreted factor, Kielin, which contains multiple cys-rich repeats similar to those in chordin (Chd). Expression of
Kielin
starts at midgastrula stages in the notochord and is detected in the floor plate of neurula embryos.
Kielin
is induced in mesoderm and in ectoderm by nodal-related genes.
Chd
is sufficient to activate
Kielin
expression in mesoderm whereas
Shh
or
HNF-3β
in addition to
Chd
is required for induction in ectoderm.
Kielin
has a distinct biological activity from that of
Chd
. Injection of
Kielin
mRNA causes dorsalization of ventral marginal zone explants and expansion of
MyoD
expression in neurula embryos. Unlike
Chd
,
Kielin
does not efficiently induce neural differentiation of animal cap ectoderm, suggesting that the activity of
Kielin
is not simply caused by BMP4 blockade. Kielin is a signaling molecule that mediates inductive activities of the embryonic midline.
Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins
Authors: Lougheed Julie C. , Holton James M. , Alber Tom , Bazan J. Fernando , Handel Tracy M.
Published: July 2002

Melanoma inhibitory activity (MIA) is a 12-kDa protein that is
secreted from both chondrocytes and malignant melanoma cells. MIA has
been reported to have effects on cell growth and adhesion, and it may
play a role in melanoma metastasis and cartilage development. We report
the 1.4-Å crystal structure of human MIA, which consists of an Src
homology 3 (SH3)-like domain with N- and C-terminal extensions of about
20 aa each. The N- and C-terminal extensions add additional structural
elements to the SH3 domain, forming a previously undescribed fold. MIA
is a representative of a recently identified family of proteins and is
the first structure of a secreted protein with an SH3 subdomain. The
structure also suggests a likely protein interaction site and suggests
that, unlike conventional SH3 domains, MIA does not recognize
polyproline helices.
Calreticulin recognizes misfolded HLA-A2 heavy chains
Authors: Mancino Laura , Rizvi Syed Monem , Lapinski Philip Edward , Raghavan Malini
Published: July 2002

Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Usingin vitrothermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits heat-induced aggregation. Inhibition of heat-induced aggregation of peptide-deficient HLA-A2 by calreticulin correlates with a rescue of the HLA-A2 heavy chain from precipitation, by forming high-molecular-weight complexes with calreticulin. Complex formation between HLA-A2 heavy chains and calreticulin occurs at 50°C but not 37°C, suggesting polypeptide-based interactions between the HLA-A2 heavy chain and calreticulin. Once complexes are formed, the addition of peptide is not sufficient to trigger efficient assembly of heavy chain/β2m/peptide complexes. Using a fluorescent peptide-based binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37°C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37°C or higher temperatures, and that oligomeric forms of calreticulin are active in inhibiting thermal aggregation of peptide-deficient HLA-A2. Taken together, these results suggest that calreticulin functions in the recognition of misfolded MHC class I heavy chains in the endoplasmic reticulum. However, in the absence of other endoplasmic reticulum components, calreticulin by itself does not enhance the assembly of misfolded MHC class I heavy chains with β2m and peptides.
Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene
Authors: Leib David A. , Machalek Michael A. , Williams Bryan R. G. , Silverman Robert H. , Virgin Herbert W.
Published: July 2002

To produce disease, viruses must enter the host, multiply locally in host tissues, spread from the site of entry, and overcome or evade host immune responses. At each stage in this infectious process, specific microbial and host genes determine the ultimate virulence of the virus. Genetic approaches have identified many viral genes that play critical roles in virulence and are presumed to target specific components of the host innate and acquired immune response. However, formal proof that a virulence gene targets a specific protein in a host pathway
in vivo
has not been obtained. Based on cell culture studies, it has been proposed that the herpes simplex virus type 1 gene ICP34.5 (ICP, infected cell protein) enhances neurovirulence by negating antiviral functions of the IFN-inducible double-stranded RNA-dependent protein kinase R or PKR [Chou, J., Chen, J.J., Gross, M. & Roizman, B. (1995)
Proc. Natl. Acad. Sci. USA
92, 10516–10520]. Herein, we show that a virus that has been attenuated by deletion of ICP34.5 exhibits wild-type replication and virulence in a host from which the PKR gene has been deleted. We show that restoration of virulence is specific to ICP34.5 and PKR by using additional host and viral mutants. The use of recombinant viruses to infect animals with null mutations in host defense genes provides a formal genetic test for identifying
in vivo
mechanisms and targets of microbial virulence genes.
Two enzymes of diacylglyceryl-
O
-4′-(
N,N,N,
-trimethyl)homoserine biosynthesis are encoded by
btaA
and
btaB
in the purple bacterium
Rhodobacter sphaeroides
Authors: Klug Rouven M. , Benning Christoph
Published: July 2002

Betaine lipids are ether-linked, nonphosphorous glycerolipids that
resemble the more commonly known phosphatidylcholine in overall
structure. Betaine lipids are abundant in many eukaryotes such as
nonseed plants, algae, fungi, and amoeba. Some of these organisms are
entirely devoid of phosphatidylcholine and, instead, contain a betaine
lipid such as
diacylglyceryl-
O
-4′-(
N,N,N,
-trimethyl)homoserine.
Recently, this lipid also was discovered in the photosynthetic purple
bacterium
Rhodobacter sphaeroides
where it seems to
replace phosphatidylcholine under phosphate-limiting growth conditions.
This discovery provided the opportunity to study the biosynthesis of
betaine lipids in a bacterial model system. Mutants of
R.
sphaeroides
deficient in the biosynthesis of the betaine lipid
were isolated, and two genes essential for this process,
btaA
and
btaB
, were identified. It is
proposed that
btaA
encodes an
S
-adenosylmethionine:diacylglycerol
3-amino-3-carboxypropyl transferase and
btaB
an
S
-adenosylmethionine-dependent
N-
methyltransferase. Both enzymatic activities can
account for all reactions of betaine lipid head group biosynthesis.
Because the equivalent reactions have been proposed for different
eukaryotes, it seems likely that orthologs of
btaA
/
btaB
may be present in other
betaine lipid-containing organisms.
Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from
Arabidopsis thaliana
define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization
Authors: Kuper Jochen , Palmer Tracy , Mendel Ralf R. , Schwarz Günter
Published: July 2002

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the enzymatic activities of molybdoenzymes. In all organisms studied so far Moco is synthesized by a unique and evolutionary old multistep pathway that requires the activities of at least six gene products. In eukaryotes, the last step of Moco synthesis, i.e., transfer and insertion of Mo into molybdopterin (MPT), is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals. Both domains (E and G) of these proteins are able to bind MPT
in vitro
. Here, we show the identification and mutational dissection of functionally important regions within the Cnx1 G domain that are essential for MPT binding, the conversion of MPT to Moco, and Moco stabilization. By functional screening for mutants in the Cnx1 G domain that are no longer able to complement
Escherichia coli
mogA
mutants, we found two classes of mutations in highly conserved amino acid residues. (
i
) The first class affects
in vitro
binding of MPT to the protein and the stabilization of Moco, the product of the G domain. (
ii
) The second class is represented by two independent mutations in the aspartate 515 position that is not affected in MPT binding and Moco stabilization; rather the conversion of MPT to Moco by using bound MPT and a yet unknown form of Mo is completely abolished. The results presented here provide biochemical evidence for a purified Cnx1 G domain catalyzing the insertion of Mo into MPT.
Estimating the age of the polydnavirus/braconid wasp symbiosis
Authors: Whitfield James B.
Published: July 2002

Polydnaviruses are essential components mediating host–parasitoid relationships between some braconid wasps and their caterpillar hosts largely by suppressing or misdirecting the host immune systems. The polydnavirus–wasp relationship is an unusual apparent mutualism between viruses and eukaryotes and remarkably has evolved to the stage where the two entities no longer can be considered separate. Estimations of the age of the polydnavirus-bearing clade of braconid wasps based on separate calculations from the mitochondrial 16S rRNA and
COI
genes and the nuclear 28S rRNA gene, calibrated using fossil data, converge to indicate a date of origin of ≈73.7 ± 10 million years ago. This range provides an upper bound on the time during which these wasps and viruses have been functionally associated.
Severe deficiencies in dopamine signaling in presymptomatic Huntington's disease mice
Authors: Bibb James A. , Yan Zhen , Svenningsson Per , Snyder Gretchen L. , Pieribone Vincent A. , Horiuchi Atsuko , Nairn Angus C. , Messer Anne , Greengard Paul
Published: July 2002

In Huntington's disease (HD), mutation of huntingtin causes selective neurodegeneration of dopaminoceptive striatal medium spiny neurons. Transgenic HD model mice that express a portion of the disease-causing form of human huntingtin develop a behavioral phenotype that suggests dysfunction of dopaminergic neurotransmission. Here we show that presymtomatic mice have severe deficiencies in dopamine signaling in the striatum. These include selective reductions in total levels of dopamine- and cAMP-regulated phosphoprotein,
M
r
32 kDA (DARPP-32) and other dopamine-regulated phosphoprotein markers of medium spiny neurons. HD mice also show defects in dopamine-regulated ion channels and in the D
1
dopamine/DARPP-32 signaling cascade. These presymptomatic defects may contribute to HD pathology.
Down-regulation of iron regulatory protein 1 gene expression by nitric oxide
Authors: Oliveira Leonor , Drapier Jean-Claude
Published: July 2002

Iron regulatory protein 1 (IRP1) is an RNA binding protein that posttranscriptionally modulates the expression of mRNAs coding for proteins involved in iron metabolism. It has long been held that its RNA binding activity is regulated posttranslationally by the insertion/extrusion of a 4Fe-4S cluster, without changes in IRP1 levels. However, the question of a possible regulation of the expression of this protein has remained open. In the present study we analyzed the modulation of IRP1 expression in murine macrophages. We showed that activation by IFN-γ and/or lipopolysaccharide, which induces IRP1 RNA binding activity via nitric oxide (NO), results simultaneously in a reduction in IRP1 protein levels, as determined by Western blot analyses. IRP1 expression decreased time-dependently to about 40% of control levels after 16 h. Down-regulation of IRP1 protein levels was correlated with the amount of NO produced and was partially abolished by the NO synthase (NOS) inhibitorN-monomethyl-l-arginine. No changes in IRP1 levels could be detected in stimulated peritoneal macrophages from NOS2 knockout (NOS2−/−) mice, unlike wild-type mice. Converse modulation of IRP1 RNA binding activity and IRP1 levels could be reproduced by exogenous NO and also was observed in nonmacrophage cells cocultured with NO-producing macrophages. We also analyzed IRP1 mRNA levels by Northern blotting and found a decrease in IRP1 mRNA expression after stimulation with IFN-γ plus lipopolysaccharide, which was abrogated in the presence ofN-monomethyl-l-arginine. This is evidence that IRP1 is regulated by a physiological stimulus other than posttranslationally.
Mechanisms that direct ordered assembly of T cell receptor β locus V, D, and J gene segments
Authors: Sleckman Barry P. , Bassing Craig H. , Hughes Maureen M. , Okada Ami , D'Auteuil Margaux , Wehrly Tara D. , Woodman Barbara B. , Davidson Laurie , Chen Jianzhu , Alt Frederick W.
Published: July 2002

T cell receptor (TCR) β variable region genes are assembled in progenitor T cells from germ-line Vβ, Dβ, and Jβ segments via an ordered two-step process in which Dβ to Jβ rearrangements occur on both alleles before appendage of a Vβ to a preexisting DJβ complex. Direct joining of Vβ segments to nonrearranged Dβ or Jβ segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCRβ locus. We have analyzed mechanisms that mediate ordered Vβ, Dβ, and Jβ assembly via an approach in which TCRβ minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that Vβ segments are preferentially targeted for rearrangement to Dβ as opposed to Jβ segments. In addition, we further demonstrated that Vβ segments can be appended to nonrearranged endogenous Dβ segments in which we have eliminated the ability of Dβ segments to join to Jβ segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.
Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation
Authors: Coffey Marcus J. , Natarajan Rama , Chumley Phillip H. , Coles Barbara , Thimmalapura Pushpa-Rekha , Nowell Mari , Kühn Hartmut , Lewis Malcolm J. , Freeman Bruce A. , O'Donnell Valerie B.
Published: July 2002

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (⋅NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed⋅NO in the presence of lipid substrate. Suppression of LOX diene conjugation by⋅NO was also found, although the lipid product profile was unchanged.⋅NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent⋅NO depletion by porcine monocytes (2.68 ± 0.03 nmol ⋅ min−1⋅ 106cells−1and 1.5 ± 0.25 nmol ⋅ min−1⋅ 106cells−1, respectively) were several-fold greater than rates of⋅NO generation by cytokine-activated macrophages (0.1–0.2 nmol ⋅ min−1⋅ 106cells−1) and LA-dependent⋅NO consumption by primary porcine monocytes inhibited⋅NO activation of soluble guanylate cyclase. These data indicate that catalytic⋅NO consumption by 12/15-LOX modulates monocyte⋅NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for⋅NO in the vasculature.
Antibodies with infinite affinity
Authors: Chmura Albert J. , Orton Molly S. , Meares Claude F.
Published: July 2002

Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs.
A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking
Authors: Ungermann Christian , Price Albert , Wickner William
Published: July 2002

The homotypic fusion of yeast vacuoles occurs in an ordered cascade of priming, docking, and fusion. The linkage between these steps has so far remained unclear. We now report that Vam7p (the vacuolar SNAP-23/25 homolog) signals from the cis-SNARE complex to Ypt7p (the vacuolar Rab/Ypt) to initiate the docking process. After Vam7p has been released from the cis-SNARE complex by Sec18p-mediated priming, it is still required for Ypt7p-dependent docking and it needs Ypt7p to remain on the vacuole. Thus, after priming, Vam7p is released from the vacuole altogether if Ypt7p has been extracted by Gdi1p or inactivated by antibody but is not released if docking is blocked simply by vacuole dilution; it is therefore Ypt7p function, and not docking per se, that retains Vam7p. In accord with this finding, cells deleted for the gene encoding Ypt7 have normal amounts of Vam7p but have little Vam7p on their isolated vacuoles. Interaction of Vam7p and Ypt7p is further indicated by two-hybrid analysis [Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P.,
et al.
(2000)
Nature (London)
403, 623–627] and by the effect of Vam7p on the association of the Rab/Ypt-effector HOPS complex (
ho
motypic fusion and vacuole
p
rotein
s
orting; Vam2p and Vam6p plus four vacuole protein sorting class C proteins) with Ypt7p. Vam7p provides a functional link between the priming step, which releases it from the cis-SNARE complex, and docking.
Femtosecond dynamics of the forbidden carotenoid S
1
state in light-harvesting complexes of purple bacteria observed after two-photon excitation
Authors: Walla Peter J. , Linden Patricia A. , Hsu Chao-Ping , Scholes Gregory D. , Fleming Graham R.
Published: July 2002

Time-resolved excited-state absorption intensities after direct
two-photon excitation of the carotenoid S
1
state are
reported for light-harvesting complexes of purple bacteria. Direct
excitation of the carotenoid S
1
state enables the
measurement of subsequent dynamics on a fs time scale without
interference from higher excited states, such as the optically allowed
S
2
state or the recently discovered dark state situated
between S
1
and S
2
. The lifetimes of the
carotenoid S
1
states in the B800-B850 complex and B800-B820
complex of
Rhodopseudomonas acidophila
are 7 ± 0.5
ps and 6 ± 0.5 ps, respectively, and in the light-harvesting
complex 2 of
Rhodobacter sphaeroides
≈1.9 ± 0.5
ps. These results explain the differences in the carotenoid to
bacteriochlorophyll energy transfer efficiency after S
2
excitation. In
Rps. acidophila
the carotenoid
S
1
to bacteriochlorophyll energy transfer is found to be
quite inefficient (φ
ET1
<28%) whereas in
Rb.
sphaeroides
this energy transfer is very efficient
(φ
ET1
≈80%). The results are rationalized by
calculations of the ensemble averaged time constants. We find that the
Car S
1
→ B800 electronic energy transfer (EET) pathway
(≈85%) dominates over Car S
1
→ B850 EET (≈15%) in
Rb. sphaeroides
, whereas in
Rps.
acidophila
the Car S
1
→ B850 EET (≈60%) is
more efficient than the Car S
1
→ B800 EET (≈40%). The
individual electronic couplings for the Car S
1
→ BChl
energy transfer are estimated to be approximately 5–26
cm
−1
. A major contribution to the difference between the
energy transfer efficiencies can be explained by different Car
S
1
energy gaps in the two species.
Distinct constitutive and low-CO
2
-induced CO
2
uptake systems in cyanobacteria: Genes involved and their phylogenetic relationship with homologous genes in other organisms
Authors: Shibata Mari , Ohkawa Hiroshi , Kaneko Takakazu , Fukuzawa Hideya , Tabata Satoshi , Kaplan Aaron , Ogawa Teruo
Published: July 2002

Cyanobacteria possess a CO
2
-concentating mechanism that
involves active CO
2
uptake and HCO

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transport. For CO
2
uptake, we have identified two systems
in the cyanobacterium
Synechocystis
sp. strain PCC 6803,
one induced at low CO
2
and one constitutive. The low
CO
2
-induced system showed higher maximal activity and
higher affinity for CO
2
than the constitutive system. On
the basis of speculation that separate NAD(P)H dehydrogenase complexes
were essential for each of these systems, we reasoned that inactivation
of one system would allow selection of mutants defective in the other.
Thus, mutants unable to grow at pH 7.0 in air were recovered after
transformation of a Δ
ndhD3
mutant with a
transposon-bearing library. Four of them had tags within
slr1302
(designated
cupB
), a homologue of
sll1734
(
cupA
), which is cotranscribed
with
ndhF3
and
ndhD3
. The
Δ
cupB,
Δ
ndhD4
, and
Δ
ndhF4
mutants showed CO
2
-uptake
characteristics of the low CO
2
induced system observed
in wild type. In contrast, mutants Δ
cupA,
Δ
ndhD3
, and Δ
ndhF3
showed
characteristics of the constitutive CO
2
-uptake system.
Double mutants impaired in one component of each of the systems were
unable to take up CO
2
and required high CO
2
for
growth. Phylogenetic analysis indicated that the
ndhD3
/
ndhD4
-,
ndhF3
/
ndhF4
-, and
cupA
/
cupB
-type genes are present only
in cyanobacteria. Most of the cyanobacterial strains studied possess
the
ndhD3
/
ndhD4-
,
ndhF3
/
ndhF4-
, and
cupA
/
cupB
-type genes in pairs. Thus,
the two types of NAD(P)H dehydrogenase complexes essential for low
CO
2
-induced and constitutive CO
2
-uptake systems
associated with the NdhD3/NdhF3/CupA-homologues and
NdhD4/NdhF4/CupB-homologues, respectively, appear to be present in
these cyanobacterial strains but not in other organisms.
Strontium isotopes reveal distant sources of architectural timber in Chaco Canyon, New Mexico
Authors: English Nathan B. , Betancourt Julio L. , Dean Jeffrey S. , Quade Jay
Published: July 2002

Between A.D. 900 and 1150, more than 200,000 conifer trees were used to build the prehistoric great houses of Chaco Canyon, New Mexico, in what is now a treeless landscape. More than one-fifth of these timbers were spruce (Picea) or fir (Abies) that were hand-carried from isolated mountaintops 75–100 km away. Because strontium from local dust, water, and underlying bedrock is incorporated by trees, specific logging sites can be identified by comparing87Sr/86Sr ratios in construction beams from different ruins and building periods to ratios in living trees from the surrounding mountains.87Sr/86Sr ratios show that the beams came from both the Chuska and San Mateo (Mount Taylor) mountains, but not from the San Pedro Mountains, which are equally close. Incorporation of logs from two sources in the same room, great house, and year suggest stockpiling and intercommunity collaboration at Chaco Canyon. The use of trees from both the Chuska and San Mateo mountains, but not from the San Pedro Mountains, as early as A.D. 974 suggests that selection of timber sources was driven more by regional socioeconomic ties than by a simple model of resource depletion with distance and time.
A role for frequenin, a Ca
2+
-binding protein, as a regulator of Kv4 K
+
-currents
Authors: Nakamura Tomoe Y. , Pountney David J. , Ozaita Ander , Nandi Sumon , Ueda Stefanie , Rudy Bernardo , Coetzee William A.
Published: July 2002

Frequenin, a Ca
2+
-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K
+
currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca
2+
-dependent manner. It also accelerates recovery from inactivation. Closely related Ca
2+
-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca
2+
-sensitive regulatory component of native A-type K
+
currents.
Nitrogen cycling by wood decomposing soft-rot fungi in the “King Midas tomb,” Gordion, Turkey
Authors: Filley Timothy R. , Blanchette Robert A. , Simpson Elizabeth , Fogel Marilyn L.
Published: July 2002

Archaeological wood in ancient tombs is found usually with extensive degradation, limiting what can be learned about the diet, environment, health, and cultural practices of the tomb builders and occupants. Within Tumulus Midas Mound at Gordion, Turkey, thought to be the tomb of the Phrygian King Midas of the 8th century B.C., we applied a stable nitrogen isotope test to infer the paleodiet of the king and determine the nitrogen sources for the fungal community that decomposed the wooden tomb, cultural objects, and human remains. Here we show through analysis of the coffin, furniture, and wooden tomb structure that the principal degrader, a soft-rot fungus, mobilized the king's highly
15
N-enriched nutrients, values indicative of a diet rich in meat, to decay wood throughout the tomb. It is also evident from the δ
15
N values of the degraded wood that the nitrogen needed for the decay of many of the artifacts in the tomb came from multiple sources, mobilized at potentially different episodes of decay. The redistribution of nutrients by the fungus was restricted by constraints imposed by the cellular structure of the different wood materials that apparently were used intentionally in the construction to minimize decay.
A tumor necrosis factor α- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase δ and proliferating cell nuclear antigen
Authors: He Hua , Tan Cheng-Keat , Downey Kathleen M. , So Antero G.
Published: July 2002

A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase δ (pol δ) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimulated pol δ activity
in vitro
in the presence, but not the absence, of PCNA, suggesting that PDIP1 also interacts functionally with both p50 and PCNA. Subcellular localization studies demonstrated that PDIP1 is a nuclear protein that colocalizes with PCNA at replication foci. A putative PCNA-binding motif was identified within the C terminus of PDIP1, and a synthetic peptide containing this PCNA-binding motif was shown to bind PCNA by far-Western analysis. Northern analysis demonstrated that PDIP1 mRNA is present in a wide variety of human tissues. PDIP1 was found to be highly homologous to a previously identified protein, B12 [Wolf, F. W., Marks, R. M., Sarma. V., Byers, M. G., Katz, R. W., Shows, T. B. & Dixit, V. M. (1992)
J. Biol. Chem.
267, 1317–1326], one of the early response genes induced by tumor necrosis factor α. PDIP1 synthesis can also be induced by tumor necrosis factor α and by IL-6, cytokines essential for liver regeneration after loss of hepatic tissue. It is suggested that PDIP1 provides a link between cytokine activation and DNA replication in liver as well as in other tissues.
Identification of five new genes on the Y chromosome of
Drosophila melanogaster
Authors: Carvalho Antonio Bernardo , Dobo Bridget A. , Vibranovski Maria D. , Clark Andrew G.
Published: July 2002

The heterochromatic state of the
Drosophila
Y chromosome has made the cloning and identification of Y-linked genes a challenging process. Here, we report application of a procedure to identify Y-linked gene fragments from the unmapped residue of the whole genome sequencing effort. Previously identified Y-linked genes appear in sequenced scaffolds as individual exons, apparently because many introns have become heterochromatic, growing to enormous size and becoming virtually unclonable. A TBLASTN search using all known proteins as query sequences, tested against a blastable database of the unmapped fragments, produced a number of matches consistent with this scenario. Reverse transcription–PCR and genetic methods were used to confirm those that are expressed, Y-linked genes. The five genes reported here include three protein phosphatases (
Pp1-Y1
,
Pp1-Y2
, and
PPr-Y
), an occludin-related gene (
ORY
), and a coiled-coils gene (
CCY
). This brings the total to nine protein-coding genes identified on the
Drosophila
Y chromosome.
ORY
and
CCY
may correspond, respectively, to the fertility factors
ks-1
and
ks-2
, whereas the three protein phosphatases represent novel genes. There remains a strong functional coherence to male function among the genes on the
Drosophila
Y chromosome.
X-ray analysis of an RNA tetraplex (UGGGGU)
4
with divalent Sr
2+
ions at subatomic resolution (0.61 Å)
Authors: Deng Junpeng , Xiong Yong , Sundaralingam Muttaiya
Published: July 2002

Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)
4
at ultra-high resolution (0.61 Å). The space group is
P
42
1
2, and cell constants are
a
=
b
= 36.16 Å and
c
= 74.09 Å. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative
br
UGGGGU and refined to 0.61-Å resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5′-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3′-end uridines are stacked-in and form uridine tetrads. All of the bases are
anti,
and the riboses are in the mixed C2′- and C3′-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight O6 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure.