bluuebunny/crossref_metadata_2025 huggingface dataset:
A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: Implications for the design of agonists and antagonists
Authors: Eckart Klaus , Jahn Olaf , Radulovic Jelena , Tezval Hossein , van Werven Lars , Spiess Joachim
Published: July 2002

In view of the observation that corticotropin-releasing factor
(CRF) affects several brain functions through at least two subtypes of
G protein-dependent receptors and a binding protein (CRFBP), we have
developed synthetic strategies to provide enhanced binding specificity.
Human/rat CRF (h/rCRF) and the CRF-like peptide sauvagine (Svg),
differing in their affinities to CRFBP by two orders of magnitude, were
used to identify the residues determining binding to CRFBP. By amino
acid exchanges, it was found that Ala
22
of h/rCRF was
responsible for this peptide's high affinity to CRFBP, whereas
Glu
21
located in the equivalent position of Svg prevented
high affinity binding to CRFBP. Accordingly,
[Glu
22
]h/rCRF was not bound with high affinity to CRFBP
in contrast to [Ala
21
]Svg, which exhibited such high
affinity. Furthermore, the affinity of both peptides to either CRF
receptor (CRFR) subtype was not reduced by these replacements, and
their subtype preference was not changed. Thus, exchange of Ala and Glu
and vice versa in positions 22 and 21 of h/rCRF and Svg,
respectively, serves as a switch discriminating between CRFBP and CRFR.
On the basis of this switch function, development of new specific CRF
agonists and antagonists is expected to be facilitated. One application
was the modification of the CRF antagonist astressin (Ast), whose
employment in animal experiments is limited by its low solubility in
cerebrospinal fluid. Introduction of Glu residues into Ast generated
with [Glu
11,16
]Ast an acidic astressin, which efficiently
antagonized
in vivo
the CRFR1-dependent reduction of
locomotion induced by ovine CRF without detectable binding to CRFBP.
Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of
Lactococcus lactis
Authors: Detmers Frank J. M. , Lanfermeijer Frank C. , Abele Rupert , Jack Ralph W. , Tampé Robert , Konings Wil N. , Poolman Bert
Published: July 2002

The oligopeptide transport system (Opp) of
Lactococcus
lactis
has the unique capacity to mediate the transport of
peptides from 4 up to at least 18 residues. The substrate specificity
of this binding protein-dependent ATP-binding cassette transporter is
determined mainly by the receptor protein OppA. To study the
specificity and ligand-binding mechanism of OppA, the following
strategy was used: (
i
) OppA was purified and anchored
via the lipid moiety to the surface of liposomes; (
ii
)
the proteoliposomes were used in a rapid filtration-based binding assay
with radiolabeled nonameric bradykinin as a reporter peptide; and
(
iii
) combinatorial peptide libraries were used to
determine the specificity and selectivity of OppA. The studies show
that (
i
) OppA is able to bind peptides up to at least 35
residues, but there is a clear optimum in affinity for nonameric
peptides; (
ii
) the specificity for nonameric peptides is
not equally distributed over the whole peptide, because positions 4, 5,
and 6 in the binding site are more selective; and (
iii
)
the differences in affinity for given side chains is relatively small,
but overall hydrophobic residues are favored—whereas glycine, proline,
and negatively charged residues lower the binding affinity. The data
indicate that not only the first six residues (enclosed by the protein)
but also the C-terminal three residues interact in a nonopportunistic
manner with (the surface of) OppA. This binding mechanism is different
from the one generally accepted for receptors of ATP-binding
cassette-transporter systems.
Impaired response to HAART in HIV-infected individuals with high autonomic nervous system activity
Authors: Cole Steve W. , Naliboff Bruce D. , Kemeny Margaret E. , Griswold Marshall P. , Fahey John L. , Zack Jerome A.
Published: July 2002

Neurotransmitters can accelerate HIV-1 replicationin vitro, leading us to examine whether differences in autonomic nervous system (ANS) activity might promote residual HIV-1 replication in patients treated with highly active antiretroviral therapy. Patients who showed constitutively high levels of ANS activity before highly active antiretroviral therapy experienced poorer suppression of plasma viral load and poorer CD4+T cell recovery over 3–11 months of therapy. ANS activity was not related to demographic or behavioral characteristics that might influence pathogenesis. However, the ANS neurotransmitter norepinephrine enhanced replication of both CCR5- and CXCR4-tropic strains of HIV-1in vitrovia chemokine receptor up-regulation and enhanced viral gene expression, suggesting that neural activity may directly promote residual viral replication.
Mammalian myotube dedifferentiation induced by newt regeneration extract
Authors: McGann Christopher J. , Odelberg Shannon J. , Keating Mark T.
Published: July 2002

Newts are capable of regenerating several anatomical structures and organs, including their limbs. This remarkable regenerative capacity is thought to depend on cellular dedifferentiation. Terminally differentiated mammalian cells, by contrast, are normally incapable of reversing the differentiation process. Several factors could explain the absence of cellular dedifferentiation in mammals: (
i
) inadequate expression of genes that initiate dedifferentiation; (
ii
) insufficient intracellular signaling pathways; (
iii
) irreversible expression of differentiation factors; and (
iv
) structural characteristics that make dedifferentiation impossible. To investigate the causes underlying the lack of cellular plasticity in mammalian cells, we examined the effect of an extract derived from newt regenerating limbs on terminally differentiated mouse C2C12 myotubes. Approximately 18% of murine myotubes reentered the cell cycle when treated with regeneration extract, whereas 25% of newt myotubes exhibited cell cycle reentry. The muscle differentiation proteins MyoD, myogenin, and troponin T were reduced to undetectable levels in 15–30% of treated murine myotubes. We observed cellular cleavage in 11% of the treated murine myotubes and approximately 50% of these myotubes continued to cleave to produce proliferating mononucleated cells. These data indicate that mammalian myotubes can dedifferentiate when stimulated with the appropriate factors and suggest that one mechanism preventing dedifferentiation of mammalian cells is inadequate spatial or temporal expression of genes that initiate dedifferentiation.
Degeneracy and complexity in biological systems
Authors: Edelman Gerald M. , Gally Joseph A.
Published: July 2002

Degeneracy, the ability of elements that are structurally different to perform the same function or yield the same output, is a well known characteristic of the genetic code and immune systems. Here, we point out that degeneracy is a ubiquitous biological property and argue that it is a feature of complexity at genetic, cellular, system, and population levels. Furthermore, it is both necessary for, and an inevitable outcome of, natural selection.
Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance
Authors: Steinman Ralph Marvin , Nussenzweig Michel C.
Published: July 2002

The immune system generally avoids horror autotoxicus or autoimmunity, an attack against the body's own constituents. This avoidance requires that self-reactive T cells be actively silenced or tolerized. We propose that dendritic cells (DCs) play a critical role in establishing tolerance, especially in the periphery, after functioning T cells have been produced in the thymus. In the steady state, meaning in the absence of acute infection and inflammation, DCs are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. Nevertheless, immature DCs continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen-loaded immature DCs silence T cells either by deleting them or by expanding regulatory T cells. This capacity of DCs to induce peripheral tolerance can work in two opposing ways in the context of infection. In acute infection, a beneficial effect should occur. The immune system would overcome the risk of developing autoimmunity and chronic inflammation if, before infection, tolerance were induced to innocuous environmental proteins as well as self antigens captured from dying infected cells. For chronic or persistent pathogens, a second but dire potential could take place. Continuous presentation of a pathogen by immature DCs, HIV-1 for example, may lead to tolerance and active evasion of protective immunity. The function of DCs in defining immunologic self provides a new focus for the study of autoimmunity and chronic immune-based diseases.
Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain
Authors: Laywell Eric D. , Rakic Pasko , Kukekov Valery G. , Holland Eric C. , Steindler Dennis A.
Published: July 2002

The mammalian brain contains a population of neural stem cells
(NSC) that can both self-renew and generate progeny along the three
lineage pathways of the central nervous system (CNS), but the
in
vivo
identification and localization of NSC in the postnatal
CNS has proved elusive. Recently, separate studies have implicated
ciliated ependymal (CE) cells, and special subependymal zone (SEZ)
astrocytes as candidates for NSC in the adult brain. In the present
study, we have examined the potential of these two NSC candidates to
form multipotent spherical clones—neurospheres—
in
vitro
. We conclude that CE cells are unipotent and give rise
only to cells within the glia cell lineage, although they are capable
of forming spherical clones when cultured in isolation. In contrast,
astrocyte monolayers from the cerebral cortex, cerebellum, spinal cord,
and SEZ can form neurospheres that give rise both to neurons and glia.
However, the ability to form neurospheres is restricted to astrocyte
monolayers derived during the first 2 postnatal wk, except for SEZ
astrocytes, which retain this capacity in the mature forebrain. We
conclude that environmental factors, simulated by certain
in
vitro
conditions, transiently confer NSC-like attributes on
astrocytes during a critical period in CNS development.
Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice
Authors: Goudy Kevin , Song Sihong , Wasserfall Clive , Zhang Y. Clare , Kapturczak Matthias , Muir Andrew , Powers Matthew , Scott-Jorgensen Marda , Campbell-Thompson Martha , Crawford James M. , Ellis Tamir M. , Flotte Terence R. , Atkinson Mark A.
Published: July 2002

The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders.
The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides
Authors: Kohli Rahul M. , Takagi Junichi , Walsh Christopher T.
Published: July 2002

Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and
N
α
-methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to αIIbβ3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.
Identification of a small molecule inhibitor of Sir2p
Authors: Bedalov Antonio , Gatbonton Tonibelle , Irvine William P. , Gottschling Daniel E. , Simon Julian A.
Published: July 2002

Sir2p is an NAD
+
-dependent histone deacetylase
required for chromatin-dependent silencing in yeast. In a cell-based
screen for inhibitors of Sir2p, we identified a compound, splitomicin,
that creates a conditional phenocopy of a
sir2
deletion mutant in
Saccharomyces cerevisiae
. Cells grown
in the presence of the drug have silencing defects at telomeres, silent
mating-type loci, and the ribosomal DNA. In addition, whole genome
microarray experiments show that splitomicin selectively inhibits
Sir2p.
In vitro
, splitomicin inhibits
NAD
+
-dependent histone deacetylase activity (HDA) of the
Sir2 protein. Mutations in
SIR2
that confer
resistance to the drug map to the likely acetylated histone tail
binding domain of the protein. By using splitomicin as a chemical
genetic probe, we demonstrate that continuous HDA of Sir2p is required
for maintaining a silenced state in nondividing cells.
Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.
Authors: Bukrinskaya A G , Ghorpade A , Heinzinger N K , Smithgall T E , Lewis R E , Stevenson M
Published: July 2002

In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.
Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.
Authors: Bouvier M , Wiley D C
Published: July 2002

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.
Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitin-dependent block of virus-like particle formation.
Authors: Menees T M , Sandmeyer S B
Published: July 2002

Many stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition.
Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.
Authors: Fisher C E , Sutherland J A , Krause J E , Murphy J R , Leeman S E , vanderSpek J C
Published: July 2002

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.
An anion channel in Arabidopsis hypocotyls activated by blue light.
Authors: Cho M H , Spalding E P
Published: July 2002

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.
Long-term potentiation at single fiber inputs to hippocampal CA1 pyramidal cells.
Authors: Isaac J T , Hjelmstad G O , Nicoll R A , Malenka R C
Published: July 2002

Despite extensive investigation, it remains unclear whether presynaptic and/or postsynaptic modifications are primarily responsible for the expression of long-term potentiation (LTP) in the CA1 region of the hippocampus. Here we address this issue by using techniques that maximize the likelihood of stimulating a single axon and thereby presumably a single synapse before and after the induction of LTP. Several basic properties of synaptic transmission were examined including the probability of neurotransmitter release (Pr), the quantal size (q), and the so-called potency, which is defined as the average size of the synaptic response when release of transmitter does occur. LTP was routinely associated with an increase in potency, whereas increases in Pr alone were not observed. LTP was also reliably induced when baseline Pr was high, indicating that synapses with high Pr can express LTP. These results suggest that the mechanism for the expression of LTP involves an increase in q and is difficult to explain by an increase in Pr alone.
Inhibition of cytokines and JAK-STAT activation by distinct signaling pathways.
Authors: Sengupta T K , Schmitt E M , Ivashkiv L B
Published: July 2002

An important component of cytokine regulation of cell growth and differentiation is rapid transcriptional activation of genes by the JAK-STAT (signal transducer and activator of transcription) signaling pathway. Ligation of cytokine receptors results in tyrosine phosphorylation and activation of receptor-associated Jak protein tyrosine kinases and cytoplasmic STAT transcription factors, which then translocate to the nucleus. We describe the interruption of cytokine triggered JAK-STAT signals by cAMP, the calcium ionophore ionomycin, and granulocyte/macrophage colony-stimulating factor. Jak1 kinase activity, interleukin 6-induced gene activation, Stat3 tyrosine phosphorylation, and DNA-binding were inhibited, as was activation of Jak1 and Stat1 by interferon gamma. The kinetics and requirement for new RNA and protein synthesis for inhibition of interleukin 6 by ionomycin and GM-CSF differed, but both agents increased the association of Jak1 with protein tyrosine phosphatase ID (SH2-containing phosphatase 2). Our results demonstrate that crosstalk with distinct signaling pathways can inhibit JAK-STAT signal transduction, and suggest approaches for modulating cytokine activity during immune responses and inflammatory processes.
Formation of mnemonic neuronal responses to visual paired associates in inferotemporal cortex is impaired by perirhinal and entorhinal lesions.
Authors: Higuchi S , Miyashita Y
Published: July 2002

Functional roles of the cortical backward signal in long-term memory formation were studied in monkeys performing a visual pair-association task. Before the monkeys learned the task, the anterior commissure was transected, disconnecting the anterior temporal cortex of each hemisphere. After training with 12 pairs of pictures, single units were recorded from the inferotemporal cortex of the monkeys as the control. By injecting a grid of ibotenic acid, we unilaterally lesioned the entorhinal and perirhinal cortex, which provides massive direct and indirect backward projections ipsilaterally to the inferotemporal cortex. After the lesion, the monkeys fixated the cue stimulus normally, relearned the preoperatively learned set (set A), and learned a new set (set B) of paired associates. Then, single units were recorded from the same area as for the prelesion control. We found that (i) in spite of the lesion, the sampled neurons responded strongly and selectively to both the set A and set B patterns and (ii) the paired associates elicited significantly correlated responses in the control neurons before the lesion but not in the cells tested after the lesion, either for set A or set B stimuli. We conclude that the ability of inferotemporal neurons to represent association between picture pairs was lost after the lesion of entorhinal and perirhinal cortex, most likely through disruption of backward neural signals to the inferotemporal neurons, while the ability of the neurons to respond to a particular visual stimulus was left intact.
Protection of nonobese diabetic mice from autoimmune diabetes by reduction of islet mass before insulitis.
Authors: Itoh A , Maki T
Published: July 2002

Nonobese diabetic mice spontaneously develop diabetes that is caused by autoimmune cell-mediated destruction of pancreatic beta cells. Here we report that surgical removal of 90% of pancreatic tissue before onset of insulitis induced a long-term diabetes-free condition in nonobese diabetic mice. Pancreatectomy after development of moderate insulitis had no effect on the course of diabetes. The effect of pancreatectomy was abrogated with subsequent development of diabetes by infusion of islet-cell-specific T lymphocytes and by transplantation of pancreatic islets. Lymphocytes from pancreatectomized diabetes-free mice exhibited low response to islet cells but responded normally to alloantigens. These results suggest that the islet cell mass plays a critical role in development of autoimmune diabetes.
Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole.
Authors: Scott S V , Hefner-Gravink A , Morano K A , Noda T , Ohsumi Y , Klionsky D J
Published: July 2002

The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.
Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.
Authors: Harter C , Pavel J , Coccia F , Draken E , Wegehingel S , Tschochner H , Wieland F
Published: July 2002

Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented.
Evolution of GABAergic circuitry in the mammalian medial geniculate body.
Authors: Winer J A , Larue D T
Published: July 2002

Many features in the mammalian sensory thalamus, such as the types of neurons, their connections, or their neurotransmitters, are conserved in evolution. We found a wide range in the proportion of gamma-aminobutyric acidergic (GABAergic) neurons in the medial geniculate body, from <1% (bat and rat) to 25% or more (cat and monkey). In the bat, some medial geniculate body subdivisions have no GABAergic cells. Species-specific variation also occurs in the somesthetic ventrobasal complex. In contrast, the lateral geniculate body of the visual system has about the same proportion of GABAergic cells in many species. In the central auditory pathway, only the medial geniculate body shows this arrangement; the relative number of GABAergic cells in the inferior colliculus and auditory cortex is similar in each species. The range in the proportion of GABAergic neurons suggests that there are comparative differences in the neural circuitry for thalamic inhibition. We conclude that the number of GABAergic neurons in thalamic sensory nuclei may have evolved independently or divergently in phylogeny. Perhaps these adaptations reflect neurobehavioral requirements for more complex, less stereotyped processing, as in speech-like communication.
Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II
Authors: Allakhverdiev S. I. , Yruela I. , Picorel R. , Klimov V. V.
Published: July 2002

It is shown that restoration of photoinduced electron flow and O
2
evolution with Mn
2+
in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0–8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H
2
CO
3
dissociation (6.4), which indicates that HCO
3
−
is the essential species for the restoration effect. The addition of just 2 Mn
2+
atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (
K
d
) of ≈2.5 μM and 20–34 μM when 2,6-dichloro-
p
-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O
2
evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the
K
d
of 20–34 μM is associated with the donor side of PSII while the location of the lower
K
d
binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.
Kinetic theory of fibrillogenesis of amyloid β-protein
Authors: Lomakin Aleksey , Teplow David B. , Kirschner Daniel A. , Benedek George B.
Published: July 2002

Prior quasielastic light scattering (QLS) studies of fibrillogenesis of synthetic amyloid β-protein (Aβ)-(1–40) at low pH have suggested a kinetic model in which: (
i
) fibrillogenesis requires a nucleation step; (
ii
) nuclei are produced by Aβ micelles in addition to seeds initially present; and (
iii
) fibril elongation occurs by irreversible binding of Aβ monomers to the fibril ends. Here we present the full mathematical formulation of this model. We describe the temporal evolution of the concentrations of Aβ monomers and micelles as well as the concentration and size distribution of fibrils. This formulation enables deduction of the fundamental parameters of the model—e.g., the nucleation and elongation rate constants
k
n
and
k
e
—from the time dependency of the apparent diffusion coefficient measured by QLS. The theory accurately represents the experimental observations for Aβ concentrations both below and above
c
*, the critical concentration for Aβ micelle formation. We suggest that the method of QLS in combination with this theory can serve as a powerful tool for understanding the molecular factors that control Aβ plaque formation.
An upper dentition of
Aframonius dieides
(Primates) from the Fayum, Egyptian Eocene
Authors: Simons Elwyn L. , Miller Ellen R.
Published: July 2002

The first known upper dentitions—an adult and subadult—of the
cercamoniine adapiform
Aframonius dieides
are described.
Comparisons show that
A. dieides
has an upper molar
morphology resembling that of other cercamoniine adapids but the
species lacks some of their typical specializations. The new dental
material confirms that
Aframonius
stands closer to
Mahgarita
from west Texas and
Cercamonius
from Europe than it does to
Schizarodon
and
Omanodon
from Oman—all of which have been ranked as
cercamoniines. Affinities of the latter two genera probably lie with
the
Anchomomys
group. The presence of a cercamoniine
adapid in the Eocene of Egypt supports the view that early African
anthropoideans evolved not in isolation, but concomitantly with a
contemporary Eocene prosimian radiation.
A micromachined device provides a new bend on fibroblast traction forces
Authors: Galbraith Catherine G. , Sheetz Michael P.
Published: July 2002

We have measured the traction forces generated by fibroblasts using a novel micromachined device that is capable of determining the subcellular forces generated by individual adhesive contacts. The front of migrating fibroblasts produced intermittent rearward forces whereas the tail produced larger forward directed forces. None of the forces were steady; they all had periodic fluctuations. The transition between forward and rearward traction forces occurred at the nucleus, not at the rear of the cell or the border between the endoplasm and the ectoplasm. We propose that the coupling of lamella extensions to fluctuating rearward tractions in front of the nuclear region move the front of a fibroblast forward, while force-facilitated release of rear adhesive contacts and anterior-directed tractions allow the region behind the nucleus to advance.
CENP-E is an essential kinetochore motor in maturing oocytes and is masked during Mos-dependent, cell cycle arrest at metaphase II
Authors: Duesbery Nick S. , Choi Taesaeng , Brown Kevin D. , Wood Kenneth W. , Resau James , Fukasawa Kenji , Cleveland Don W. , Vande Woude George F.
Published: July 2002

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from
mos
-deficient (MOS
−/−
) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.
Identification of a structural constituent and one possible site of postembryonic formation of a teleost otolithic membrane
Authors: Davis James G. , Burns Frank R. , Navaratnam Dasakumar , Lee A. Masaji , Ichimiya Shingo , Oberholtzer J. Carl , Greene Mark I.
Published: July 2002

A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (
Lepomis macrochirus
) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein
in vivo
using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein,
in vivo
, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.
Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability
Authors: Scalley Michelle L. , Baker David
Published: July 2002

The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of
1/T
on constant stability contours in the temperature–denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.
A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor α chain
Authors: Wang Yonghong , Shen Bo-Jiang , Sebald Walter
Published: July 2002

Human interleukin 4 (IL-4) binds to its cellular receptor with a
K
d
in the subnanomolar range, similar to many other 4-helix-bundle proteins interacting with members of the hematopoietin (cytokine) receptor superfamily. In the IL-4 system this interaction is predominantly determined by the extracellular domain (IL4-BP) of the receptor α chain (
K
d
≈ 150 pM). Now a high-resolution mutational and kinetic analysis has revealed that the high-affinity binding of IL-4 originates from a continuous patch of a few mostly polar or charged amino acid side chains located on helices A and C. The binding epitope comprises (
i
) a set of side chains determining the dissociation rate (
k
off
) and (
ii
) a partially overlapping set determining the association rate constant (
k
on
) of the IL-4/IL4-BP complex. The
k
off
epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance. The cumulative increase in
k
off
after alanine substitution is 10
5
-fold for the central mixed-charge pair and 3 × 10
3
-fold for the satellites. The
k
on
epitope is formed by five positively charged residues on helix C (Lys-77, Arg-81, Lys-84, Arg-85, and Arg-88) and two neighboring residues on helix A (Glu-9 and Thr-13). The cumulative loss in
k
on
of the alanine variants is only about 10-fold. These results provide the basis for an understanding of molecular recognition in cytokine receptor complexes and for an IL-4 antagonist design.
Four highly stable eukaryotic mRNAs assemble 3′ untranslated region RNA–protein complexes sharing cis and trans components
Authors: Holcik Martin , Liebhaber Stephen A.
Published: July 2002

Assembly of a sequence-specific RNA–protein complex on the 3′ untranslated region (3′UTR) of human α-globin mRNA (α-complex) correlates with mRNA stabilization. Here we map a limited segment of the α-globin 3′UTR that is both necessary and sufficient for α-complex formation. The sequence of this binding region identifies three additional, highly stable mRNAs that share closely related, pyrimidine-rich cis-motifs in their respective 3′UTRs. Each mRNA assembles a sequence-specific ribonucleoprotein complex at this conserved region. These complexes are structurally related, and each contains a 39-kDa cytoplasmic poly(C) binding protein previously demonstrated to be essential to formation of the α-complex. These observations indicate the existence of a general determinant for stabilization of eukaryotic mRNAs.
The role of temporal cortical areas in perceptual organization
Authors: Sheinberg D. L. , Logothetis N. K.
Published: July 2002

The visual areas of the temporal lobe of the primate are thought to be essential for the representation of visual objects. To examine the role of these areas in the visual awareness of a stimulus, we recorded the activity of single neurons in monkeys trained to report their percepts when viewing ambiguous stimuli. Visual ambiguity was induced by presenting incongruent images to the two eyes, a stimulation condition known to instigate binocular rivalry, during which one image is seen at a given time while the other is perceptually suppressed. Previous recordings in areas V1, V2, V4, and MT of monkeys experiencing binocular rivalry showed that only a small proportion of striate and early extrastriate neurons discharge exclusively when the driving stimulus is seen. In contrast, the activity of almost all neurons in the inferior temporal cortex and the visual areas of the cortex of superior temporal sulcus was found to be contingent upon the perceptual dominance of an effective visual stimulus. These areas thus appear to represent a stage of processing beyond the resolution of ambiguities—and thus beyond the processes of perceptual grouping and image segmentation—where neural activity reflects the brain’s internal view of objects, rather than the effects of the retinal stimulus on cells encoding simple visual features or shape primitives.
Marginal fitness contributions of nonessential genes in yeast
Authors: Thatcher J. W. , Shaw Janet M. , Dickinson W. J.
Published: July 2002

Analysis of the complete genome sequence of
Saccharomyces cerevisiae
confirms and extends earlier evidence that a majority of yeast genes are not essential, at least under laboratory conditions. Many fail to yield a discernible mutant phenotype even when disrupted. Genes not subject to natural selection would accumulate inactivating mutations, so these “cryptic” genes must have functions that are overlooked by the standard methods of yeast genetics. Two explanations seem possible: (
i
) They have important functions only in environments not yet duplicated in the laboratory and would have conditional phenotypes if tested appropriately. (
ii
) They make small, but significant, contributions to fitness even under routine growth conditions, but the effects are not large enough to be detected by conventional methods. We have tested the second “marginal benefit” hypothesis by measuring the fitnesses of a random collection of disruption mutants in direct competition with their wild-type progenitor. A substantial majority of mutant strains that lack obvious defects nevertheless are at a significant selective disadvantage just growing on rich medium under normal conditions. This result has important implications for efforts to understand the functions of novel genes revealed by sequencing projects.
Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion
Authors: Nakayama Y. , Kobayashi I.
Published: July 2002

We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing. Such selfish behavior may have contributed to the spread and maintenance of RM systems. Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes. We identified the C gene of
Eco
RV as a positive regulator of restriction. A C mutation eliminated postsegregational killing by
Eco
RV. The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity. Consistent with this proposal, bacteria preexpressing
ecoRVC
were transformed at a reduced efficiency by plasmids carrying the
Eco
RV RM gene complex. Cells carrying the
Bam
HI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a
Pvu
II RM gene complex, which shares the same C specificity. The reduction most likely was caused by chromosome cleavage at unmodified
Pvu
II sites by prematurely expressed
Pvu
II restriction enzyme. Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex. This phenomenon, termed “apoptotic mutual exclusion,” is reminiscent of suicidal defense against virus infection programmed by other selfish elements.
pvuIIC
and
bamHIC
genes define one incompatibility group of exclusion whereas
ecoRVC
gene defines another.