bluuebunny/crossref_metadata_2025 huggingface dataset:
A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase
Authors: Sträter Norbert , Sun Lee , Kantrowitz E. R. , Lipscomb William N.
Published: July 2002

The active sites of aminopeptidase A (PepA) from
Escherichia coli
and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when
l
-leucine
p
-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces
k
cat
/
K
m
about 10,000-fold.
An internal targeting signal directing proteins into the mitochondrial intermembrane space
Authors: Diekert Kerstin , Kispal Gyula , Guiard Bernard , Lill Roland
Published: July 2002

Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis. Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage. Mitochondrial heme lyases lack N-terminal targeting information. These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to
c
type cytochromes. For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient. Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins. The targeting sequence is highly conserved among all known heme lyases. Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues. These features clearly distinguish this signal from classical presequences. When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis. The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information. It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space.
A model of the detection of warmth and cold by cutaneous sensors through effects on voltage-gated membrane channels
Authors: Adair Robert K.
Published: July 2002

Warmth and cold sensations are known to derive from separate warm and cold cutaneous thermoreceptors in the form of differentiated afferent nerves. The firing rate of warm-sensing nerves increases as the temperature increases; the firing rate of cold-sensing nerves increases if the temperature is reduced. I postulate that the primary sensitivity of the warm sensors derives from voltage-gated Ca
2+
membrane channels configured such that an increase in temperature opens channels and increases the ion influx while a reduction in temperature increases the ion influx through voltage-gated Na
+
channels in the cold sensory nerve ends. In either case, the initial cation influx causes a small cellular depolarization that further opens Ca
2+
channels, admitting more cations in a positive feedback process that leads to the depolarization of the membrane, thus initiating an action potential pulse. Monte Carlo calculations based on a well defined model of such processes, which include effects of noise, demonstrate quantitative agreement of the model with an extensive body of data.
A protein phosphatase 2C gene,
LjNPP2C1
, from
Lotus japonicus
induced during root nodule development
Authors: Kapranov Philipp , Jensen Trine Juul , Poulsen Carsten , de Bruijn Frans J. , Szczyglowski Krzysztof
Published: July 2002

Symbiotic interactions between legumes and compatible strains of
rhizobia result in root nodule formation. This new plant organ provides
the unique physiological environment required for symbiotic nitrogen
fixation by the bacterial endosymbiont and assimilation of this
nitrogen by the plant partner. We have isolated two related genes
(
LjNPP2C1
and
LjPP2C2
) from the model
legume
Lotus japonicus
that encode protein phosphatase
type 2C (PP2C). Expression of the
LjNPP2C1
gene was
found to be enhanced specifically in
L. japonicus
nodules, whereas the
LjPP2C2
gene was expressed at a
similar level in nodules and roots
.
A glutathione
S
-transferase–LjNPP2C1 fusion protein was shown to have
Mg
2+
- or Mn
2+
-dependent and okadaic
acid-insensitive PP2C activity
in vitro
. A chimeric
construct containing the full-length LjNPP2C1 cDNA, under the control
of the
Saccharomyces cerevisiae
alcohol dehydrogenase
promoter, was found to be able to complement a yeast PP2C-deficient
mutant (
pct1Δ)
. The transcript level of the
LjNPP2C1
gene was found to increase significantly in
mature nodules, and its highest expression level occurred after
leghemoglobin (
lb
) gene induction, a molecular marker
for late developmental events in nodule organogenesis. Expression of
the
LjNPP2C1
gene was found to be drastically altered in
specific
L. japonicus
lines carrying monogenic-recessive
mutations in symbiosis-related loci, suggesting that the product of the
LjNPP2C1
gene may function at both early and late stages
of nodule development.
Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons
Authors: Narisawa-Saito Mako , Silva Alcino J. , Yamaguchi Tsuyoshi , Hayashi Takashi , Yamamoto Tadashi , Nawa Hiroyuki
Published: July 2002

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant
in vivo
reduction in these AMPA receptor proteins but not in their mRNA levels.
In vitro
kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.
Correlation of regional cerebral blood flow and change of plasma sodium concentration during genesis and satiation of thirst
Authors: Denton Derek , Shade Robert , Zamarippa Frank , Egan Gary , Blair-West John , McKinley Michael , Fox Peter
Published: July 2002

Positron emission tomography studies were conducted during genesis of moderate thirst by rapid i.v. infusion of hypertonic saline (0.51 M) and after satiation of thirst by drinking water. The correlation of regional cerebral blood flow with the change in the plasma Na concentration showed a significant group of cerebral activations in the anterior cingulate region and also a site in the middle temporal gyrus and in the periaqueductal gray. Strongest deactivations occurred in the parahippocampal and frontal gyri. The data are consistent with an important role of the anterior cingulate in the genesis of thirst.
The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol
Authors: Munday A. D. , Norris F. A. , Caldwell K. K. , Brown S. , Majerus P. W. , Mitchell C. A.
Published: July 2002

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)
P
2
]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)
P
2
production.
ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes
Authors: Zhu Yunxiang , Drake Matthew T. , Kornfeld Stuart
Published: July 2002

The assembly of clathrin-coated vesicles on Golgi membranes is initiated by the GTP-binding protein ADP ribosylation factor (ARF), which generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex. Once bound, the AP-1 recruits clathrin triskelia, which polymerize to form the coat. We have found that ARF⋅GTP also recruits AP-1 and clathrin onto protein-free liposomes. The efficiency of this process is modulated by the composition of the liposomes, with phosphatidylserine being the most stimulatory phospholipid. There is also a requirement for cytosolic factor(s) other than ARF. Thin-section electron microscopy shows the presence of clathrin-coated buds and vesicles that resemble those formed
in vivo
. These results indicate that AP-1-containing clathrin-coated vesicles can form in the absence of integral membrane proteins. Thus, ARF⋅GTP, appropriate lipids, and cytosolic factor(s) are the minimal components necessary for AP-1 clathrin-coat assembly.
Genetic complexity of pathogen perception by plants: The example of
Rcr3
, a tomato gene required specifically by
Cf-2
Authors: Dixon Mark S. , Golstein Catherine , Thomas Colwyn M. , van der Biezen Erik A. , Jones Jonathan D. G.
Published: July 2002

Genetic analysis of plant–pathogen interactions has
demonstrated that resistance to infection is often determined by the
interaction of dominant plant resistance (
R
) genes and
dominant pathogen-encoded avirulence (
Avr
) genes. It was
postulated that
R
genes encode receptors for Avr
determinants. A large number of
R
genes and their
cognate
Avr
genes have now been analyzed at the
molecular level.
R
gene loci are extremely polymorphic,
particularly in sequences encoding amino acids of the leucine-rich
repeat motif. A major challenge is to determine how Avr perception by R
proteins triggers the plant defense response. Mutational analysis has
identified several genes required for the function of specific R
proteins. Here we report the identification of
Rcr3
, a
tomato gene required specifically for
Cf-2-
mediated
resistance. We propose that Avr products interact with host proteins to
promote disease, and that R proteins “guard” these host
components and initiate Avr-dependent plant defense responses.
Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to
Pseudomonas aeruginosa
infections
Authors: Pier Gerald B.
Published: July 2002

Chronic
Pseudomonas aeruginosa
infection occurs in
75–90% of patients with cystic fibrosis (CF). It is the foremost
factor in pulmonary function decline and early mortality. A connection
has been made between mutant or missing CF transmembrane conductance
regulator (CFTR) in lung epithelial cell membranes and a failure in
innate immunity leading to initiation of
P. aeruginosa
infection. Epithelial cells use CFTR as a receptor for internalization
of
P. aeruginosa
via endocytosis and subsequent removal
of bacteria from the airway. In the absence of functional CFTR, this
interaction does not occur, allowing for increased bacterial loads in
the lungs. Binding occurs between the outer core of the bacterial
lipopolysaccharide and amino acids 108–117 in the first predicted
extracellular domain of CFTR. In experimentally infected mice,
inhibiting CFTR-mediated endocytosis of
P. aeruginosa
by
inclusion in the bacterial inoculum of either free bacterial
lipopolysaccharide or CFTR peptide 108–117 resulted in increased
bacterial counts in the lungs. CFTR is also a receptor on
gastrointestinal epithelial cells for
Salmonella
enterica
serovar Typhi, the etiologic agent of typhoid fever.
There was a significant decrease in translocation of this organism to
the gastrointestinal submucosa in transgenic mice that are heterozygous
carriers of a mutant ΔF508
CFTR
allele, suggesting
heterozygous
CFTR
carriers may have increased resistance
to typhoid fever. The identification of CFTR as a receptor for
bacterial pathogens could underlie the biology of CF lung disease and
be the basis for the heterozygote advantage for carriers of mutant
alleles of
CFTR
.
Intentionality detection and “mindreading”: Why does game form matter?
Authors: McCabe Kevin A. , Smith Vernon L. , LePore Michael
Published: July 2002

By around the age of 4 years, children “can work out what
people might know, think or believe” based on what they say or do.
This is called “mindreading,” which builds upon the human ability
to infer the intentions of others. Game theory makes a strong
assumption about what individual A can expect about B's intentions and
vice versa
, viz. that each is a self-interested opponent
of the other and will reliably analyze games by using such basic
principles as dominance and backward induction, and behave as if the
normal form of an extensive form game is equivalent to the latter. But
the extensive form allows intentions to be detected from actual
sequential play and is therefore not necessarily equivalent
psychologically to the normal form. We discuss Baron-Cohen's theory of
the mindreading system [Baron-Cohen, S. (1995)
Mindblindness:
An Essay on Autism and Theory of Mind
(MIT Press, Cambridge,
MA)] to motivate the comparison of behavior in an extensive form game
with its corresponding normal form. As in the work of Rapoport
[Rapoport, A. (1997)
Int. J. Game Theory
26, 113–136]
and Schotter
et al.
[Schotter, A., Wiegelt, K. &
Wilson, C. (1994)
Games Econ. Behav.
6,
445–468], we find consistent differences in behavior between the
normal and extensive forms. In particular, we observe attempts to
cooperate, and in some treatments we observe the achievement of
cooperation, occurring more frequently in the extensive form.
Cooperation in this context requires reciprocity, which is more
difficult to achieve by means of intentionality detection in the normal
as opposed to the extensive form games we study.
Geoelectric potential changes: Possible precursors to earthquakes in Japan
Authors: Uyeda S. , Nagao T. , Orihara Y. , Yamaguchi T. , Takahashi I.
Published: July 2002

Whether electromagnetic precursors to earthquakes (EQs) exist is an important question not only for EQ prediction but also for understanding the physical processes of EQ generation. Slow transient geoelectric potential changes have been observed before several recent EQs in Japan. In most cases, they appeared 1–19 days before the EQs, and their duration and intensity were several minutes to 1 h and 1–2 mV/100 m. The changes appeared before five of all six EQs with magnitude ≥ 5 that occurred within 20 km of our stations during the observation period. Changes were also detected at greater epicentral distances (up to 75 km) before two other EQs, including one EQ of magnitude 4.7, which was preceded by a signal simultaneously recorded at three widely separated stations. These geoelectric potential changes have been distinguished through the following criteria from a multitude of other changes, which were noise of various origins. (
i
) The selected changes were proportional in amplitude to the length of the recording station's short (≈100 m) dipoles and were simultaneously detected also on long (1–10 km) dipoles when the latter were in operation. (
ii
) No such changes occurred during the observation period that were not followed by EQs. Although the EQ precursory nature of these geoelectric potential changes is admittedly unproven, it seems that the present results warrant continued serious research into the occurrence, generation, and transmission of these signals and their possible causal relationship to EQs.
Polymorphism of dopamine D2 and D4 receptor genes and Slavic‐surnamed alcoholic patients
Authors: OVCHINNIKOV IGOR V. , DRUZINA EKATERINA , OVTCHINNIKOVA OLGA , ZAGOROVSKAYA TATIANA , NEBARAKOVA TAMARA , ANOKHINA IRINA P.
Published: July 2002

The association between alcoholism and the Taq1 “A” and “B” polymorphic alleles at the DRD2 gene and 48‐bp tandem repeat in exon 3 of dopamine D4 receptor (DRD4) gene in 42 unrelated Slavic‐surnamed patients and 76 normal controls was examined. The frequency of the A1 allele was higher in alcoholic patients and in alcoholic patients with a family history of alcoholism than in controls (χ2= 3.45, p < 0.001 and χ2= 3.97, p < 0.001, respectively). Moreover, the frequency of the A1 allele was higher in alcoholics with a family history of alcoholism than in alcoholics without a family history (χ2= 3.33, p < 0.001).The results of association analysis for both the Taq1 “B” and DRD4 alleles were negative for alcoholics in general, subgroups of alcoholics and normal controls. However, the 7‐repeat allele (DRD4*7R) of DRD4 gene occurred at significantly higher frequency in alcoholics with a family history of alcoholism compared with those without a family history (χ2= 3.42, p < 0.01).The results indicate that the A1 allele of the DRD2 gene is associated with susceptibility to alcoholism in general.The A1 allele, as well as the DRD4*7R allele, is significantly prevalent among alcoholics with a family history, in comparison with alcoholics without a family history, reflecting different roles of genetic factors in development of alcoholism.
Expression of ZntA, a Zinc‐Transporting P1‐Type ATPase, is Specifically Regulated by Zinc and Cadmium
Authors: Noll Meenakshi , Lutsenko Svetlana
Published: July 2002

All cells have developed various mechanisms to regulate precisely the availability of important micronutrients such as zinc and copper; in many cells, this regulation is mediated by P‐type ATPases. Most of the P1‐ATPases have been described very recently, and little is known about their molecular mechanism and regulation. Here, we demonstrate that the expression of ZntA, a Zn,Cd‐transporting P‐type ATPase of
Phosphorylation of Human Pro-Urokinase on Ser138/303 Impairs Its Receptor-dependent Ability to Promote Myelomonocytic Adherence and Motility
Authors: Franco Paola , Iaccarino Ciro , Chiaradonna Ferdinando , Brandazza Anna , Iavarone Carlo , Mastronicola M. Rosaria , Nolli M. Luisa , Stoppelli M. Patrizia
Published: July 2002

Serine phosphorylation of human pro-urokinase (pro-uPA) by A431 human carcinoma cells results in a catalytically active molecule with reduced sensitivity to plasminogen activator inhibitor type 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered prouPA variants carrying a COOH-terminal poly-histidine tag. Stably transfected A431 cells do not incorporate radioactive phosphate into tagged pro-uPA in which the serines 138 and 303 have been replaced with glutamic residues, although endogenous nontagged pro-uPA is 32P-labeled on A and B chains. Moreover, the catalyticindependent ability of the mono- and di-substituted “phosphorylation-like” variants to bind to the GPIanchored urokinase receptor (uPAR) and promote adherence of differentiating U937, HL-60, and THP-1 myelomonocytic cells was examined. We found that glutamic residues as well as the naturally occurring phosphoserines at positions 138 and 303 abolish proadhesive ability, although they do not interfere with receptor binding. In addition, pro-uPA carrying Glu138/303 lacks the capability to induce a chemotactic response of THP-1 cells. The exclusive presence of Glu138 reduces pro-uPA proadhesive and chemotactic ability by 70– 80%, indicating that a phosphoserine residue at the same position plays a major inhibitory role of myeloid cell response to pro-urokinase. The di-substitution does not affect pro-uPA ability to interact with vitronectin or to enhance binding of urea-denatured vitronectin to uPAR. However, unlike wild-type tagged pro-uPA, the di-substituted variant does not induce receptor polarization in pre-adherent U937 cells. Taken together, the data support the possibility that pro-uPA phosphorylation on Ser138/303 can modulate uPAR transducing ability.
The Effect of Apical and Basolateral Lipids on the Function of the Band 3 Anion Exchange Protein
Authors: Hof Wouter van't , Malik Abha , Vijayakumar S. , Qiao Jizeng , Adelsberg Janet van , Al-Awqati Qais
Published: July 2002

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type–specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein.

We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM1 (Gal 1β1, 3GalNAcβ1, 4Gal-NeuAcα2, 3Galβ1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4,4′-dinitorsostilbene-2,2′-disulfonic acid (DNDS; a reversible stilbene) on 35SO4/SO4 exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM1 reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.
Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen
Authors: Shapiro Paul S. , Vaisberg Eugeni , Hunt Alan J. , Tolwinski Nicholas S. , Whalen Anne M. , McIntosh J. Richard , Ahn Natalie G.
Published: July 2002

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal–regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311–1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.
The Eps15 Homology (Eh) Domain-Based Interaction between Eps15 and Hrb Connects the Molecular Machinery of Endocytosis to That of Nucleocytosolic Transport
Authors: Doria Margherita , Salcini Anna Elisabetta , Colombo Emanuela , Parslow Tristram G. , Pelicci Pier Giuseppe , Di Fiore Pier Paolo
Published: July 2002

The Eps15 homology (EH) module is a protein–protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15–Hrb complex in regulating the stability of Rev.
Identification of Epi64, a Tbc/Rabgap Domain–Containing Microvillar Protein That Binds to the First PDZ Domain of Ebp50 and E3karp
Authors: Reczek David , Bretscher Anthony
Published: July 2002

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1–like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50–PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain–containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.
Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether
Authors: Dwir Oren , Kansas Geoffrey S. , Alon Ronen
Published: July 2002

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein α-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin–mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin–carbohydrate bond.
Cation–chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes
Authors: Strick Reiner , Strissel Pamela L. , Gavrilov Konstantin , Levi-Setti Riccardo
Published: July 2002

Mammalian interphase and mitotic cells were analyzed for their cation composition using a three-dimensional high resolution scanning ion microprobe. This instrument maps the distribution of bound and unbound cations by secondary ion mass spectrometry (SIMS). SIMS analysis of cryofractured interphase and mitotic cells revealed a cell cycle dynamics of Ca2+, Mg2+, Na+, and K+. Direct analytical images showed that all four, but no other cations, were detected on mitotic chromosomes. SIMS measurements of the total cation content for diploid chromosomes imply that one Ca2+ binds to every 12.5–20 nucleotides and one Mg2+ to every 20–30 nucleotides. Only Ca2+ was enriched at the chromosomal DNA axis and colocalized with topoisomerase IIα (Topo II) and scaffold protein II (ScII). Cells depleted of Ca2+ and Mg2+ showed partially decondensed chromosomes and a loss of Topo II and ScII, but not hCAP-C and histones. The Ca2+-induced inhibition of Topo II catalytic activity and direct binding of Ca2+ to Topo II by a fluorescent filter-binding assay supports a regulatory role of Ca2+ during mitosis in promoting solely the structural function of Topo II. Our study directly implicates Ca2+, Mg2+, Na+, and K+ in higher order chromosome structure through electrostatic neutralization and a functional interaction with nonhistone proteins.
A Novel Mechanism for B Cell Repertoire Maturation Based on Response by B Cell Precursors to Pre–B Receptor Assembly
Authors: Wasserman R. , Li Y.-S. , Shinton S.A. , Carmack C.E. , Manser T. , Wiest D.L. , Hayakawa K. , Hardy R.R.
Published: July 2002

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin μ heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre–B cell progression as in adult bone marrow, this complex inhibits pre–B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 μ heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.
Aminooxypentane-RANTES Induces CCR5 Internalization but Inhibits Recycling: A Novel Inhibitory Mechanism of HIV Infectivity
Authors: Mack Matthias , Luckow Bruno , Nelson Peter J. , Cihak Josef , Simmons Graham , Clapham Paul R. , Signoret Nathalie , Marsh Mark , Stangassinger Manfred , Borlat Fréderic , Wells Timothy N.C. , Schlöndorff Detlef , Proudfoot Amanda E.I.
Published: July 2002

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.
One of the CD3ε Subunits within a T Cell Receptor Complex Lies in Close Proximity to the Cβ FG Loop
Authors: Ghendler Yoseph , Smolyar Alex , Chang Hsiu-Ching , Reinherz Ellis L.
Published: July 2002

A recent crystal structure of the N15 α/β-T cell receptor (TCR) in complex with an Fab derived from the H57 Cβ-specific monoclonal antibody (mAb) shows the mAb fragment interacting with the elongated FG loop of the Cβ domain. This loop creates one side wall of a cavity within the TCR Ti-α/β constant region module (CαCβ) while the CD and EF loops of the Cα domain form another wall. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3ε ectodomain. By using specific mAbs to mouse TCR-β (H57) and CD3ε (2C11) subunits, we herein provide evidence that only one of the two CD3ε chains within the TCR complex is located in close proximity to the TCR Cβ FG loop, in support of the above notion. Moreover, analysis of T cells isolated from transgenic mice expressing both human and mouse CD3ε genes shows that the heterologous human CD3ε component can replace the mouse CD3ε at this site. The location of one CD3ε subunit within the rigid constant domain module has implications for the mechanism of signal transduction throughout T cell development.
Interleukin (IL)-4–independent Induction of Immunoglobulin (Ig)E, and Perturbation of  T Cell Development in Transgenic Mice Expressing IL-13
Authors: Emson Claire L. , Bell Sarah E. , Jones Alison , Wisden William , McKenzie Andrew N.J.
Published: July 2002

Recent studies using interleukin (IL)-4–deficient animals have highlighted the existence of IL-4–independent immunoglobulin (Ig)E induction. We have established transgenic mice expressing IL-13 from a transgene comprising a genomic fragment containing the IL-13 gene and the human CD2 locus control region. The transgenes were expressed in lymphoid tissues and induced by T cell activators, suggesting regulation by elements of the IL-13 promoter. IL-13 transgenic lines expressed 10–100-fold higher levels of serum IgE than their littermate controls, but had normal levels of other serum Ig isotypes. Elevated IgE levels were also detected in sera from IL-4–deficient mice carrying IL-13 transgenes, indicating that IL-4 is not required for IL-13–induced IgE expression in the mouse. Expression of IL-13 also perturbed the development of thymocytes. Although thymocyte development was normal up to 4 wk of age, thymocyte number decreased dramatically thereafter, reaching 10% of normal by 10 wk, and despite normal size and appearance, histological examination demonstrated that transgenic thymi contained only small foci of thymocytes. The reduction in thymocyte number was due mainly to a depletion of CD4+CD8+ thymocytes, and did not affect significantly the composition of peripheral T cell populations. These data indicate that expression of IL-13 transgenes in vivo can regulate IgE production in the mouse, and that IL-13 may also influence thymocyte development.
Syk Tyrosine Kinase and B Cell Antigen Receptor (BCR) Immunoglobulin-α Subunit Determine BCR-mediated Major Histocompatibility Complex Class II–restricted Antigen Presentation
Authors: Lankar Danielle , Briken Volker , Adler Kristin , Weiser Peter , Cassard Sylvanie , Blank Ulrich , Viguier Mireille , Bonnerot Christian
Published: July 2002

Stimulation of CD4+ helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-α/Ig-β heterodimers which, second, target antigens to MHC class II–containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-α–associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-α cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide– MHC class II complexes through antigen targeting by BCR subunits.
Plasmodium falciparum Produces Prostaglandins that are Pyrogenic, Somnogenic, and Immunosuppressive Substances in Humans
Authors: Kilunga Kubata B. , Eguchi Naomi , Urade Yoshihiro , Yamashita Kouwa , Mitamura Toshihide , Tai Kumiko , Hayaishi Osamu , Horii Toshihiro
Published: July 2002

Plasmodium falciparum causes the most severe form of human malaria, which kills ∼1.5–2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host–parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2α. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography–selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.
Cd40–Cd40 Ligand Interactions in Vivo Regulate Migration of Antigen-Bearing Dendritic Cells from the Skin to Draining Lymph Nodes
Authors: Moodycliffe Angus M. , Shreedhar Vijay , Ullrich Stephen E. , Walterscheid Jeffrey , Bucana Corazon , Kripke Margaret L. , Flores-Romo Leopoldo
Published: July 2002

Whereas CD40–CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand −/− mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand −/− mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand −/− mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-α production and was corrected by injecting recombinant TNF-α or an agonistic anti-CD40 monoclonal antibody. Thus, CD40–CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-α production and play a vital role in the initiation of acquired T cell–mediated immunity.
H-2D Ligand Expression by Ly49A+ Natural Killer (NK) Cells Precludes Ligand Uptake from Environmental Cells
Authors: Zimmer Jacques , Ioannidis Vassilios , Held Werner
Published: July 2002

To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2Dd using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for Dd, Ly49A, were specifically underrepresented among cells with low Dd levels. That was due to the acquisition of Dd molecules by the Ly49A+ NK cells that have lost their Dd transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were Dd+, uptake of the alternative ligand Dk was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on Dd+ NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2bDd as compared with H-2b NK cells, rendering Ly49A+ NK cells in Dd+ mice particularly reactive. Minor reductions of Dd levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by Dd on the same cell via a lateral binding site in the H-2Dd molecule.
Identification of Dynamically Distinct Subpopulations of T Lymphocytes That Are Differentially Affected by HIV
Authors: Kovacs Joseph A. , Lempicki Richard A. , Sidorov Igor A. , Adelsberger Joseph W. , Herpin Betsey , Metcalf Julia A. , Sereti Irini , Polis Michael A. , Davey Richard T. , Tavel Jorge , Falloon Judith , Stevens Randy , Lambert Laurie , Dewar Robin , Schwartzentruber Douglas J. , Anver Miriam R. , Baseler Michael W. , Masur Henry , Dimitrov Dimiter S. , Lane H. Clifford
Published: July 2002

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = −0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.