bluuebunny/crossref_metadata_2025 huggingface dataset:
Global properties of the mapping between local amino acid sequence and local structure in proteins.
Authors: Han K F , Baker D
Published: July 2002

Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence.
Identification of interdomain sequences promoting the intronless evolution of a bacterial protein family.
Authors: de Château M , Björck L
Published: July 2002

In the evolution of eukaryotic genes, introns are believed to have played a major role in increasing the probability of favorable duplication events, chance recombinations, and exon shuffling resulting in functional hybrid proteins. As a rule, prokaryotic genes lack introns, and the examples of prokaryotic introns described do not seem to have contributed to gene evolution by exon shuffling. Still, certain protein families in modern bacteria evolve rapidly by recombination of genes, duplication of functional domains, and as shown for protein PAB of the anaerobic bacterial species Peptostreptococcus magnus, by the shuffling of an albumin-binding protein module from group C and G streptococci. Characterization of a protein PAB-related gene in a P. magnus strain with less albumin-binding activity revealed that the shuffled module was missing. Based on this fact and observations made when comparing gene sequences of this family of bacterial surface proteins interacting with albumin and/or immunoglobulin, a model is presented that can explain how this rapid intronless evolution takes place. A new kind of genetic element is introduced: the recer sequence promoting interdomain, in frame recombination and acting as a structure-less flexibility-promoting spacer in the corresponding protein. The data presented also suggest that antibiotics could represent the selective pressure behind the shuffling of protein modules in P. magnus, a member of the indigenous bacterial flora.
Induction of centromeric activity in maize by suppressor of meiotic drive 1.
Authors: Dawe R K , Cande W Z
Published: July 2002

The Abnormal chromosome 10 (Ab10) in maize causes normally-quiescent blocks of heterochromatin called knobs to function as meiotic centromeres. Under these circumstances genetic markers associated with knobs exhibit meiotic drive, i.e., they are preferentially transmitted to progeny. Here we describe a mutation called suppressor of meiotic drive (smd1) that partially suppresses meiotic drive, and demonstrate that smd1 causes a quantitative reduction in the mobility of knobs on the meiotic spindle. We conclude that Smd1 encodes a product that is necessary for the activation of ectopic centromeres, and that meiotic drive occurs as a consequence of the resulting change in chromosome movement. As a genetic system, Ab10 offers a new and powerful approach for analyzing centromere/kinetochore function.
Degradation of CYC1 mRNA in the yeast Saccharomyces cerevisiae does not require translation.
Authors: Yun D F , Sherman F
Published: July 2002

Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYC1 alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The cyc1-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYC1 mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYC1 mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5'-->3'. Also, the increased stability of CYC1 mRNAs in xrn1-delta strains lacking Xrn1p, the major 5'-->3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrn1p, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYC1 mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway.
On the mechanism of action of ribonuclease A: relevance of enzymatic studies with a p-nitrophenylphosphate ester and a thiophosphate ester.
Authors: Breslow R , Chapman W H
Published: July 2002

It has been reported that His-119 of ribonuclease A plays a major role as an imidazolium ion acid catalyst in the cyclization/cleavage of normal dinucleotides but that it is not needed for the cyclization/cleavage of 3'-uridyl p-nitrophenyl phosphate. We see that this is also true for simple buffer catalysis, where imidazole (as in His-12 of the enzyme), but not imidazolium ion, plays a significant catalytic role with the nitrophenyl substrate, but both are catalytic for normal dinucleotides such as uridyluridine. Rate studies show that the enzyme catalyzes the cyclization of the nitrophenylphosphate derivative 47,000,000 times less effectively (kcat/kuncat) than it does uridyladenosine, indicating that approximately 50% of the catalytic free energy change is lost with this substrate. This suggests that the nitrophenyl substrate is not correctly bound to take full advantage of the catalytic groups of the enzyme and is thus not a good guide to the mechanism used by normal nucleotides. The published data on kinetic effects with ribonuclease A of substituting thiophosphate groups for the phosphate groups of normal substrates has been discussed elsewhere, and it was argued that these effects are suggestive of the classical mechanism for ribonuclease action, not the novel mechanism we have recently proposed. The details of these rate effects, including stereochemical preferences in the thiophosphate series, can be invoked as support for our newer mechanism.
Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily.
Authors: Griffith D L , Keck P C , Sampath T K , Rueger D C , Carlson W D
Published: July 2002

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.
Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.
Authors: Brown P K , Curtiss R
Published: July 2002

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.
Bootstrap confidence levels for phylogenetic trees
Authors: Efron Bradley , Halloran Elizabeth , Holmes Susan
Published: July 2002

Evolutionary trees are often estimated from DNA or RNA sequence
data. How much confidence should we have in the estimated trees? In
1985, Felsenstein [Felsenstein, J. (1985)
Evolution
39,
783–791] suggested the use of the bootstrap to answer this question.
Felsenstein’s method, which in concept is a straightforward
application of the bootstrap, is widely used, but has been criticized
as biased in the genetics literature. This paper concerns the use of
the bootstrap in the tree problem. We show that Felsenstein’s method
is not biased, but that it can be corrected to better agree with
standard ideas of confidence levels and hypothesis testing. These
corrections can be made by using the more elaborate bootstrap method
presented here, at the expense of considerably more computation.
Site-specific crosslinking of mammalian U11 and U6atac to the 5′ splice site of an AT–AC intron
Authors: Yu Yi-Tao , Steitz Joan A.
Published: July 2002

A rare class of introns with AT–AC at their termini recently has been identified in metazoan genes. Splicing of these introns requires a different set of small nuclear ribonucleoprotein particles (snRNPs) (U11, U12, U5, and U4atac/U6atac) compared with the snRNPs (U1, U2, U5, and U4/U6) required for splicing the majority of pre-mRNA introns, but otherwise little is known regarding the excision of AT–AC introns. Here we use site-specific 4-thiouridine (
4S
U) crosslinking analysis to dissect the mechanism of 5′ splice site recognition during
in vitro
splicing of the AT–AC intron from the P120 pre-mRNA. Upon irradiation with 365-nm UV light, three P120 substrates, each with a single
4S
U substitution near the 5′ splice site (at position +2, +4, or +7), produce two early ATP-independent crosslinks with similar kinetics. For one of the substrates, P120-
4S
U+2, a third ATP-requiring crosslink forms as the two early crosslinks diminish. RNase H digestion coupled with Northern blotting indicates that the two early crosslinks generated with P120-
4S
U+2 contain the U11 small nuclear RNA. Reverse transcription–PCR followed by cloning and sequencing demonstrates that the third crosslink involves U6atac. The dynamic appearance of the three crosslinks correlates with the kinetics of the splicing reaction and suggests that the 5′ splice site is recognized first by U11 and then by U6atac. Our results argue that the splicing of AT–AC introns is mechanistically similar to the splicing of the major class of introns and that the U11 and U6atac snRNPs in the AT–AC spliceosome fulfill analogous roles to U1 and U6, respectively, in the major spliceosome.
Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity
Authors: Wagner Paul D. , Steeg Patricia S. , Vu Ngoc-Diep
Published: July 2002

Nm23
genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type
nm23-H1
, but not by transfections with two
nm23-H1
mutants,
nm23-H1

S12OG

and
nm23-H1

P96S

. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1
P96S
or nm23-H1
S120G
, and another was phosphorylated much more slowly by nm23-H1
P96S
and by nm23-H1
S120G
than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1
S120G
and nm23-H1
P96S
were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1
S120
was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1
P96S
was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.
RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels
Authors: Doupnik Craig A. , Davidson Norman , Lester Henry A. , Kofuji Paulo
Published: July 2002

G protein-gated inward rectifier K+(GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.
Down-regulation of interferon γ-activated STAT1 by UV light
Authors: Aragane Yoshinori , Kulms Dagmar , Luger Thomas A. , Schwarz Thomas
Published: July 2002

STAT1 is a cytoplasmic transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon-γ (IFNγ). Phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFNγ-inducible genes. Here, we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby hindering IFNγ from exerting its biological effects. This effect is not due to a down-regulation of the IFNγ receptor because phosphorylation of upstream-located Jak1 and Jak2 was not suppressed by UV light. In contrast, UV light had no effect on the phosphorylation of STAT3, which is activated by the proinflammatory cytokine interleukin 6. The UV light effect on STAT1 phosphorylation could be antagonized by vanadate, indicating at least partial involvement of a protein tyrosine phosphatase. Therefore, this study indicates a mechanism by which UV light can inhibit gene activation and suggests STAT1 as a new extranuclear UV target closely located to the membrane.
Loss of haloperidol induced gene expression and catalepsy in protein kinase A-deficient mice
Authors: Adams Monique R. , Brandon Eugene P. , Chartoff Elena H. , Idzerda Rejean L. , Dorsa Daniel M. , McKnight G. Stanley
Published: July 2002

The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-
fos
mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIβ subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIβ mutant mice fail to induce either c-
fos
or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.
Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein
Authors: Huang Jianing , Gao Xiong , Li Shyun , Cao Zhaodan
Published: July 2002

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.
The restriction–modification genes of
Escherichia coli
K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities
Authors: O’Neill Mary , Chen Angela , Murray Noreen E.
Published: July 2002

Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system
Eco
KI does not behave in the same way. The genes specifying
Eco
KI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If
Eco
KI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993)
Mol. Gen. Genet.
241, 491–496] to be relevant to control of expression of the genes that encode
Eco
KI. Our evidence suggests that not all R-M systems are evolving as “selfish” units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.
Site-directed mutations reveal long-range compensatory interactions in the
Adh
gene of
Drosophila melanogaster
Authors: Parsch John , Tanda Soichi , Stephan Wolfgang
Published: July 2002

Long-range interactions between the 5′ and 3′ ends of mRNA molecules have been suggested to play a role in the initiation of translation and the regulation of gene expression. To identify such interactions and to study their molecular evolution, we used phylogenetic analysis to generate a model of mRNA higher-order structure in the
Adh
transcript of
Drosophila melanogaster
. This model predicts long-range, tertiary contacts between a region of the protein-encoding sequence just downstream of the start codon and a conserved sequence in the 3′ untranslated region (UTR). To further examine the proposed structure, site-directed mutations were generated
in vitro
in a cloned
D. melanogaster Adh
gene, and the mutant constructs were introduced into the
Drosophila
germ line through P-element mediated transformation. Transformants were spectrophotometrically assayed for alcohol dehydrogenase activity. Our results indicate that transformants containing a silent mutation near the start of the protein-encoding sequence show an ≈15% reduction in alcohol dehydrogenase activity relative to wild-type transformants. This activity can be restored to wild-type levels by a second, compensatory mutation in the 3′ UTR. These observations are consistent with a higher-order structure model that includes long-range interactions between the 5′ and 3′ ends of the
Adh
mRNA. However, our results do not fit the classical compensatory substitution model because the second mutation by itself (in the 3′ UTR) did not show a measurable reduction in gene expression.
Subunit arrangement in the human 20S proteasome
Authors: Kopp Friedrich , Hendil Klavs B. , Dahlmann Burkhardt , Kristensen Poul , Sobek Axel , Uerkvitz Wolfgang
Published: July 2002

In human 20S proteasomes two copies of each of seven different α-type and seven different β-type subunits are assembled to form a stack of four seven-membered rings, giving the general structure α1–7, β1–7, β1–7, α1–7. By means of immunoelectron microscopy and chemical crosslinking of neighboring subunits, we have determined the positions of the individual subunits in the proteasome. The topography shows that for the trypsin-like, the chymotrypsin-like, and the postglutamyl cleaving activities, the pairs of β type subunits, which are thought to form active sites, are nearest neighbors.
Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins
Authors: Miesenböck Gero , Rothman James E.
Published: July 2002

The emission of light, coupled to exocytosis, can in principle be utilized to monitor the activity of a large number of individual synapses simultaneously. To illustrate this concept, fusion proteins ofCypridinaluciferase and synaptotagmin-I or VAMP-2/synaptobrevin (which we term “synaptolucins”) were expressed in cultured hippocampal neurons with the help of viral vectors. Synaptolucins were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with their cognate luciferin, which was added to the extracellular medium. Photon emissions required a depolarizing stimulus, occurred from regions with high synaptic density as ascertained by vital staining of recycling synaptic vesicles, and were sensitive to Ca2+depletion and clostridial neurotoxins. The method can currently detect exocytosis of the readily releasable pool of synaptic vesicles at a hippocampal synapse, corresponding to about two dozen quanta, but has the potential for greater sensitivity.
Plasma membrane localization is required for RGS4 function in
Saccharomyces cerevisiae
Authors: Srinivasa Sreesha P. , Bernstein Leah S. , Blumer Kendall J. , Linder Maurine E.
Published: July 2002

RGS4, a mammalian GTPase activating protein for G protein α
subunits, was identified by its ability to inhibit the pheromone
response pathway in
Saccharomyces cerevisiae
. To define
regions of RGS4 necessary for its function
in vivo
, we
assayed mutants for activity in this system. Deletion of the N-terminal
33 aa of RGS4 (Δ1–33) yielded a nonfunctional protein and loss of
plasma membrane localization. These functions were restored by addition
of a C-terminal membrane-targeting sequence to RGS4 (Δ1–33). Thus,
plasma membrane localization is tightly coupled with the ability of
RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to
green fluorescent protein was sufficient to localize an otherwise
soluble protein to the plasma membrane, defining this N-terminal region
as a plasma membrane anchorage domain. RGS4 is palmitoylated, with
Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly,
mutation of the cysteine residues within the N-terminal domain of RGS4
did not affect plasma membrane localization in yeast or the ability to
inhibit signaling. Features of the N-terminal domain other than
palmitoylation are responsible for the plasma membrane association of
RGS4 and its ability to inhibit pheromone response in yeast.
Adenosine acts by A
1
receptors to stimulate release of prolactin from anterior-pituitaries
in
vitro
Authors: Yu Wen H. , Kimura Mayumi , Walczewska Anna , Porter John C. , McCann Samuel M.
Published: July 2002

Adenosine has been identified in the anterior pituitary gland and is secreted from cultured folliculostellate (FS) cells. To determine whether adenosine controls the secretion of anterior pituitary hormones
in vitro
, adenosine was incubated with anterior pituitaries. It stimulated prolactin (PRL) release at the lowest concentration used (10
−10
M); the stimulation peaked at 10
−8
M with a threefold increase in release and declined to minimal stimulation at 10
−4
and 10
−3
M. Follicle-stimulating hormone release was maximally inhibited at 10
−8
M, whereas luteinizing hormone release was not significantly inhibited. Two selective A
1
adenosine receptor antagonists (10
−7
or 10
−5
M) had no effect on basal PRL release, but either antagonist completely blocked the response to the most effective concentration of adenosine (10
−8
M). In contrast, a highly specific A
2
receptor antagonist (10
−7
or 10
−5
M) had no effect on basal PRL release or the stimulation of PRL release induced by adenosine (10
−8
M). We conclude that adenosine acts to stimulate PRL release
in vitro
by activating A
1
receptors. Since the A
1
receptors decrease intracellular-free calcium, this would decrease the activation of nitric oxide synthase in the FS cells, resulting in decreased release of nitric oxide (NO). NO inhibits PRL release by activating guanylate cyclase that synthesizes cGMP from GTP; cGMP concentrations increase in the lactotrophs leading to inhibition of PRL release. In the case of adenosine, NO release from the FS cells decreases, resulting in decreased concentrations of NO in the lactotrophs, consequent decreased cGMP formation, and resultant increased PRL release.
Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import
Authors: Fransen Marc , Terlecky Stanley R. , Subramani Suresh
Published: July 2002

The discovery of many fatal human disorders resulting from impaired peroxisomal protein import makes the functional characterization of human peroxins critical. As part of our attempt to identify novel human genes and gene products involved in the import of peroxisomal proteins, we raised antisera against peroxisomal membrane proteins. One such antiserum inhibited peroxisomal protein import in semipermeabilized mammalian cells. This “import inhibiting” antiserum, ab-MF3, specifically recognized a 57-kDa protein. Immunoblot analysis of rat liver subcellular fractions demonstrated that this protein was present exclusively in peroxisomal membranes. Functional analysis revealed that this 57-kDa molecule bound the PTS1 receptor, Pex5p, in ligand blots, suggesting it is a docking site on the peroxisomal membrane. Previous studies have identified two yeast proteins, Pex14p and Pex13p, as Pex5p-binding proteins. To facilitate the biochemical analysis of peroxisomal membrane docking proteins, we cloned and expressed the previously unidentified human Pex14p, as well as a human Pex13p that is 39 aa longer than previously reported. Recombinant Pex14p was specifically recognized by the “import inhibiting” ab-MF3 and bound Pex5p and the Src homology 3 (SH3) domain of Pex13p in ligand blots. These studies demonstrate that the ab-MF3-immunoreactive, 57-kDa peroxisomal membrane protein is Pex14p. Furthermore, this peroxin interacts with Pex5p and Pex13p(SH3) and is directly required for peroxisomal protein import.
The human
GNAS1
gene is imprinted and encodes distinct paternally and biallelically expressed G proteins
Authors: Hayward Bruce E. , Kamiya Mamoru , Strain Lisa , Moran Veronica , Campbell Roderick , Hayashizaki Yoshihide , Bonthron David T.
Published: July 2002

The
GNAS1
gene encodes the α subunit of the G protein G
s
, which couples receptor binding by several hormones to activation of adenylate cyclase. Null mutations of
GNAS1
cause pseudohypoparathyroidism (PHP) type Ia, in which hormone resistance occurs in association with a characteristic osteodystrophy. The observation that PHP Ia almost always is inherited maternally has led to the suggestion that
GNAS1
may be an imprinted gene. Here, we show that, although G
s
α expression (directed by the promoter upstream of exon 1) is biallelic,
GNAS1
is indeed imprinted in a promoter-specific fashion. We used parthenogenetic lymphocyte DNA to screen by restriction landmark genomic scanning for loci showing differential methylation between paternal and maternal alleles. This screen identified a region that was found to be methylated exclusively on a maternal allele and was located ≈35 kb upstream of
GNAS1
exon 1. This region contains three novel exons that are spliced into alternative
GNAS1
mRNA species, including one exon that encodes the human homologue of the large G protein XLαs. Transcription of these novel mRNAs is exclusively from the paternal allele in all tissues examined. The differential imprinting of separate protein products of
GNAS1
therefore may contribute to the anomalous inheritance of PHP Ia.
The adaptive nature of the human neurocognitive architecture: An alternative model
Authors: Cerra Peggy La , Bingham Roger
Published: July 2002

The model of the human neurocognitive architecture proposed by evolutionary psychologists is based on the presumption that the demands of hunter-gatherer life generated a vast array of cognitive adaptations. Here we present an alternative model. We argue that the problems inherent in the biological markets of ancestral hominids and their mammalian predecessors would have required an adaptively flexible, on-line information-processing system, and would have driven the evolution of a functionally plastic neural substrate, the neocortex, rather than a confederation of evolutionarily prespecified social cognitive adaptations. In alignment with recent neuroscientific evidence, we suggest that human cognitive processes result from the activation of constructed cortical representational networks, which reflect probabilistic relationships between sensory inputs, behavioral responses, and adaptive outcomes. The developmental construction and experiential modification of these networks are mediated by subcortical circuitries that are responsive to the life history regulatory system. As a consequence, these networks are intrinsically adaptively constrained. The theoretical and research implications of this alternative evolutionary model are discussed.
Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin
Authors: Menozzi Franco D. , Bischoff Rainer , Fort Emmanuelle , Brennan Michael J. , Locht Camille
Published: July 2002

Although it generally is accepted that the interaction ofMycobacterium tuberculosiswith alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned fromM. tuberculosisand bacillus Calmette–Guérin, and the sequence was found to be identical between the two species. The calculatedMrwas smaller than the observedMrwhen analyzed by SDS/PAGE. This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety. Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates. Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein. Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway.
Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water
Authors: Schmitke Jennifer L. , Stern Lawrence J. , Klibanov Alexander M.
Published: July 2002

The x-ray crystal structures of
trans
-cinnamoyl–subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl–subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.
Polyazamacrolides from ladybird beetles: Ring-size selective oligomerization
Authors: Schroeder Frank C. , Smedley Scott R. , Gibbons Leah K. , Farmer Jay J. , Attygalle Athula B. , Eisner Thomas , Meinwald Jerrold
Published: July 2002

The pupal defensive secretion of the 24-pointed ladybird beetle,
Subcoccinella vigintiquatuorpunctata
, consists of a mixture of macrocyclic polyamines, dominated by the three dimeric, 30-membered macrocycles 11-13, derived from the two building blocks 11-(2-hydoxyethylamino)-5-tetradecenoic acid (9) and 11-(2-hydoxyethylamino)-5,8-tetradecadienoic acid (10). Smaller amounts of the four possible cyclic trimers of 9 and 10 were also detected, corresponding to 45-membered macrocycles. Structural assignments were based on NMR-spectroscopic investigations and HPLC–MS analyses. In addition, the all-
S
absolute configuration of the
S. vigintiquatuorpunctata
macrocycles was determined by comparison of derivatives of the natural material with enantiomerically pure synthetic samples. Comparing this alkaloid mixture with that of the pupal defensive secretion in related ladybird beetle species indicates that the degree of oligomerization of the 2-hydroxyethylamino carboxylic acid building blocks can be carefully controlled by the insects.
Modes of action of aspirin-like drugs: Salicylates inhibit Erk activation and integrin-dependent neutrophil adhesion
Authors: Pillinger Michael H. , Capodici Constance , Rosenthal Pamela , Kheterpal Neil , Hanft Simon , Philips Mark R. , Weissmann Gerald
Published: July 2002

The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness offormylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1–8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion byformylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.
Classification of hypergeometric identities for π and other logarithms of algebraic numbers
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: July 2002

This paper provides transcendental and algebraic framework for the classification of identities expressing π and other logarithms of algebraic numbers as rapidly convergent generalized hypergeometric series in rational parameters. Algebraic and arithmetic relations between values of

p
+1

F
p

hypergeometric functions and their values are analyzed. The existing identities are explained, and new exhaustive classes of new ones are presented.
High-resolution restriction maps of bacterial artificial chromosomes constructed by optical mapping
Authors: Cai Weiwen , Jing Junping , Irvin Benjamin , Ohler Lynne , Rose Elise , Shizuya Hiroaki , Kim Ung-Jim , Simon Melvin , Anantharaman Thomas , Mishra Bhubaneswar , Schwartz David C.
Published: July 2002

Large insert clone libraries have been the primary resource used for the physical mapping of the human genome. Research directions in the genome community now are shifting direction from purely mapping to large-scale sequencing, which in turn, require new standards to be met by physical maps and large insert libraries. Bacterial artificial chromosome libraries offer enormous potential as the chosen substrate for both mapping and sequencing studies. Physical mapping, however, has come under some scrutiny as being “redundant” in the age of large-scale automated sequencing. We report the development and applications of nonelectrophoretic, optical approaches for high-resolution mapping of bacterial artificial chromosome that offer the potential to complement and thereby advance large-scale sequencing projects.
DNA sequence of the mitochondrial hypervariable region II from the Neandertal type specimen
Authors: Krings Matthias , Geisert Helga , Schmitz Ralf W. , Krainitzki Heike , Pääbo Svante
Published: July 2002

The DNA sequence of the second hypervariable region of the mitochondrial control region of the Neandertal type specimen, found in 1856 in central Europe, has been determined from 92 clones derived from eight overlapping amplifications performed from four independent extracts. When the reconstructed sequence is analyzed together with the previously determined DNA sequence from the first hypervariable region, the Neandertal mtDNA is found to fall outside a phylogenetic tree relating the mtDNAs of contemporary humans. The date of divergence between the mtDNAs of the Neandertal and contemporary humans is estimated to 465,000 years before the present, with confidence limits of 317,000 and 741,000 years. Taken together, the results support the concept that the Neandertal mtDNA evolved separately from that of modern humans for a substantial amount of time and lends no support to the idea that they contributed mtDNA to contemporary modern humans.
Molecular genetics of ecological diversification: Duplication and rapid evolution of toxin genes of the venomous gastropod
Conus
Authors: Duda Thomas F. , Palumbi Stephen R.
Published: July 2002

Predatory snails in the marine gastropod genus
Conus
stun prey by injecting a complex mixture of peptide neurotoxins. These conotoxins are associated with trophic diversification and block a diverse array of ion channels and neuronal receptors in prey species, but the evolutionary genesis of this functional diversity is unknown. Here we show that conotoxins with little amino acid similarity are in fact products of recently diverged loci that are rapidly evolving by strong positive selection in the vermivorous cone,
Conus abbreviatus
, and that the rate of conotoxin evolution is higher than that of most other known proteins. Gene duplication and diversifying selection result in the formation of functionally variable conotoxins that are linked to ecological diversification and evolutionary success of this genus.
The tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein kinase Akt/PKB in B lymphocytes
Authors: Li Hsiu-Ling , Davis William W. , Whiteman Eileen L. , Birnbaum Morris J. , Puré Ellen
Published: July 2002

The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals.
Clonal dynamics of progressive neoplastic transformation
Authors: Chow Ming , Rubin Harry
Published: July 2002

In a recent study, we found that newly isolated clones of NIH 3T3 mouse cells undergo neoplastic transformation more readily than uncloned cultures from which they were derived. After eleven low-density passages (LDPs), most of the 29 clones produced lightly stained early-stage transformed foci when grown to confluence in a primary assay for transformation, and one of them consistently produced a few tiny dense foci. In the present work, six of the clones were kept in LDPs for 56 passages and assayed for focus formation at confluence at six passage levels. The clone that produced tiny dense foci switched to light foci during the LDPs, four others produced light foci at different passage levels, and one progressed from light to dense foci after the last passage. By contrast, all the clones progressed to dense focus formation in five or fewer serial repetitions of the assay at confluence. Because all but one of the clones underwent about half as many total divisions at each LDP as they did when grown to the stationary state at confluence, the latter is more efficient in eliciting progression than the exponential growth of the LDPs. Extension of the period at confluence of uncloned cultures results in the appearance of dense foci within light foci. Because the latter are localized clonal populations, the intrafocal progression reinforces the conclusion that clonal expansion favors transformation. We discuss the significance of these results for the clonal origin of human cancer and the increased incidence of cancer with age.
Sugars modulate an unusual mode of control of the cell-wall invertase gene (
Incw
1) through its 3′ untranslated region in a cell suspension culture of maize
Authors: Cheng Wan-Hsing , Taliercio Earl W. , Chourey Prem S.
Published: July 2002

We show here that a cell-wall invertase encoded by the
Incw
1 gene is regulated at both the transcriptional and posttranscriptional levels by sugars in a heterotrophic cell suspension culture of maize. The
Incw
1 gene encoded two transcripts:
Incw
1-S (small) and
Incw
1-L (large); the size variation was attributable to different lengths in the 3′ untranslated region. Both metabolizable and nonmetabolizable sugars induced
Incw
1-L RNA apparently by default. However, only the metabolizable sugars, sucrose and
d
-glucose, were associated with the increased steady-state abundance of
Incw
1-S RNA, the concomitant increased levels of INCW1 protein and enzyme activity, and the downstream metabolic repression of the sucrose synthase gene,
Sh
1. Conversely, nonmetabolizable sugars, including the two glucose analogs 3-
O
-methylglucose and 2-deoxyglucose, induced greater steady-state levels of the
Incw
1-L RNA, but this increase did not lead to either an increase in the levels of the INCW1 protein/enzyme activity or the repression of the
Sh
1 gene. We conclude that sugar sensing and the induction of the
Incw
1 gene is independent of the hexokinase pathway. More importantly, our results also suggest that the 3′ untranslated region of the
Incw
1 gene acts as a regulatory sensor of carbon starvation and may constitute a link between sink metabolism and cellular translation in plants.