bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal trefoil factor induces inactivation of extracellular signal-regulated protein kinase in intestinal epithelial cells
Authors: Kanai Michiyuki , Mullen Colleen , Podolsky Daniel K.
Published: July 2002

Intestinal trefoil factor (ITF), a small, compact protease-resistant peptide, is abundantly expressed in goblet cells of large and small intestine. Although several biological activities of ITF have been identified, including promotion of wound healing, stimulation of epithelial cell migration, and protection of intestinal epithelial barrier, little is known about signaling events through which ITF mediates its physiological function. In this study, the effects of exogenous ITF on mitogen-activated protein kinase (MAPK) signaling cascades were examined in IEC-6 cells, a nontransformed intestinal epithelial cell line that does not express endogenous trefoil peptides. Stimulation with ITF resulted in rapid decrease in extracellular signal-related protein kinase (ERK) activity and concomitant reduced ERK tyrosine phosphorylation. ITF also decreased activation of ERK activity induced by either transforming growth factor-α, which links extracellular stimuli to the Ras/Raf/MEK/ERK pathway via the epidermal growth factor receptor, or phorbol 12-myristate 13-acetate, which activates Raf through protein kinase C. ITF-induced inhibition of ERK activity was blocked by an inhibitor of tyrosine and dual-specific phosphatases, sodium orthovanadate. In summary, ITF leads to inhibition of ERK and the MAPK pathway through activation of tyrosine or dual-specific phosphatase.
A test of the background selection hypothesis based on nucleotide data fromDrosophila ananassae
Authors: Stephan Wolfgang , Xing Lin , Kirby David A. , Braverman John M.
Published: July 2002

We estimated DNA sequence variation within and between four populations ofDrosophila ananassaeatOm(1D)andvermilion(v) by using single-strand conformation polymorphism analysis and direct DNA sequencing.Om(1D)is located on the X chromosome in a region with a normal recombination rate;vis in a region of low recombination. In each population, levels of nucleotide diversity atvare reduced 10- to 25-fold relative to those atOm(1D). Divergence betweenD. ananassaeand its sibling speciesD. pallidosa, however, is comparable for both loci. This lack of correlation between levels of polymorphism and divergence led to the rejection of a constant-rate, neutral model. To distinguish among alternative models, we propose a test of the background selection hypothesis based on the observed pattern of differentiation between populations. Although the degree of differentiation (measured byFST) among all pairs of subpopulations is similar atOm(1D), we found substantial differences atv. The two northern populations from Burma and Nepal are very homogeneous, whereas comparisons between northern and southern populations (e.g., between Nepal and middle India) produced largeFSTvalues. A coalescent-based simulation of the background selection model (in a geographically structured species with a finite number of demes) showed that the observed homogeneity among the northern populations is inconsistent with the background selection hypothesis. Instead, it may have been caused by a recent hitchhiking event that was limited to the northern species range.
Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)
n
Authors: Dobi A. , v. Agoston D.
Published: July 2002

Submillimolar levels of calcium, similar to the physiological total (bound + free) intranuclear concentration (0.01–1 mM), induced a conformational change within d(TG/AC)
n
, one of the frequent dinucleotide repeats of the mammalian genome. This change is calcium-specific, because no other tested cation induced it and it was detected as a concentration-dependent transition from B- to a non-B-DNA conformation expanding from 3′ end toward the 5′ of the repeat. Genomic footprinting of various rat brain regions revealed the existence of similar non-B-DNA conformation within a d(TG/AC)
28
repeat of the endogenous enkephalin gene only in enkephalin-expressing caudate nucleus and not in the nonexpressing thalamus. Binding assays demonstrated that DNA could bind calcium and can compete with calmodulin for calcium.
Transcytosis of α
1
-acidic glycoprotein in the continuous microvascular endothelium
Authors: Predescu D. , Predescu S. , McQuistan T. , Palade G. E.
Published: July 2002

By using perfusions and bolus administration, coupled with postembedding immunocytochemical procedures, we have identified the structures involved in the transport of derivatized orosomucoid (α
1
-acidic glycoprotein) across the continuous microvascular endothelium of the murine myocardium. Our findings indicate that: (
i
) monomeric orosomucoid binds to the luminal surface of the endothelium; (
ii
) it is restricted to caveolae during its transport across the endothelium; (
iii
) it is detected in the perivascular spaces at early time points (by 1 min) and in larger quantities at later time points (>5 min) from the beginning of its perfusion or its intravascular administration; (
iv
) no orosomucoid molecules are found in the intercellular junctions or at the abluminal exits of interendothelial spaces; and (
v
) the vesicular transport of orosomucoid is strongly inhibited by
N
-ethylmaleimide (>80%). Because, by size and shape, the orosomucoid qualifies as a preferential probe for the postulated small pore system, our results are discussed in relation to the pore theory of capillary permeability.
Effects of Zn
2+
on wild and mutant neuronal α
7
nicotinic receptors
Authors: Palma E. , Maggi L. , Miledi R. , Eusebi F.
Published: July 2002

Zn
2+
is a key structural/functional component of many proteins and is present at high concentrations in the brain and retina, where it modulates ligand-gated receptors. Therefore, a study was made of the effects of zinc on homomeric neuronal nicotinic receptors expressed in
Xenopus
oocytes after injection of cDNAs encoding the chicken wild or mutant α
7
subunits. In oocytes expressing wild-type receptors, Zn
2+
alone did not elicit appreciable membrane currents. Acetylcholine (AcCho) elicited large currents (
I
AcCho
) that were reduced by Zn
2+
in a reversible and dose-dependent manner, with an IC
50
of 27 μM and a Hill coefficient of 0.4. The inhibition of
I
AcCho
by Zn
2+
was competitive and voltage-independent, a behavior incompatible with a channel blockade mechanism. In sharp contrast, in oocytes expressing a receptor mutant, with a threonine-for-leucine 247 substitution (
L247T
α
7
), subnanomolar concentrations of Zn
2+
elicited membrane currents (
I
Zn
) that were reversibly inhibited by the nicotinic receptor blockers methyllycaconitine and α-bungarotoxin. Cell-attached single-channel recordings showed that Zn
2+
opened channels that had a mean open time of 5 ms and a conductance of 48 pS. At millimolar concentrations Zn
2+
reduced
I
AcCho
and the block became stronger with cell hyperpolarization. Thus, Zn
2+
is a reversible blocker of wild-type α
7
receptors, but becomes an agonist, as well as an antagonist, following mutation of the highly conserved leucine residue 247 located in the M2 channel domain. We conclude that Zn
2+
is a modulator as well as an activator of homomeric nicotinic α
7
receptors.
Inhibition of human telomerase by 2′-
O
-methyl-RNA
Authors: Pitts Anne E. , Corey David R.
Published: July 2002

Telomerase, a ribonucleoprotein up-regulated in many types of cancers, possesses an RNA template necessary to bind and extend telomere ends. The intrinsic accessibility of telomerase to incoming nucleic acids makes the RNA template an ideal target for inhibition by oligonucleotides. We report here that 2′-
O
-methyl-RNA (2′-
O
-meRNA), an oligonucleotide chemistry known to exert sequence-specific effects in cell culture and animals, inhibits telomerase with potencies superior to those possessed by analogous peptide nucleic acids (PNAs). Potent inhibition relative to PNAs is surprising, because the binding affinity of 2′-
O
-meRNAs for complementary RNA is low relative to analogous PNAs. A 2′-
O
-meRNA oligomer with terminal phosphorothioate substitutions inhibits telomerase sequence-selectively within human-tumor-derived DU145 cells when delivered with cationic lipids. In contrast to the ability of 2′-
O
-meRNA oligomers to inhibit telomerase, the binding of a 2′-
O
-meRNA to an inverted repeat within plasmid DNA was not detectable, whereas binding of PNA was efficient, suggesting that the relative accessibility of the telomerase RNA template is essential for inhibition by 2′-
O
-meRNA. Inhibition of telomerase by 2′-
O
-meRNA will facilitate probing the link between telomerase activity and sustained cell proliferation and may provide a basis for the development of chemopreventive and chemotherapeutic agents.
Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N
Authors: Nohturfft Axel , Brown Michael S. , Goldstein Joseph L.
Published: July 2002

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.
High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids
Authors: Wand A. Joshua , Ehrhardt Mark R. , Flynn Peter F.
Published: July 2002

The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.
Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology
Authors: Cohenford Menashi A. , Rigas and Basil
Published: July 2002

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (
i
) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (
ii
) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.
Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized
Authors: Lauffenburger Douglas A. , Oehrtman Gregory T. , Walker Laura , Wiley H. Steven
Published: July 2002

Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.
A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish
Authors: Ginns Edward I. , St. Jean Pamela , Philibert Robert A. , Galdzicka Marzena , Damschroder-Williams Patricia , Thiel Bonnie , Long Robert T. , Ingraham Loring J. , Dalwaldi Harnisha , Murray Melissa A. , Ehlert Melissa , Paul Sharon , Remortel Brian G. , Patel Ashima P. , Anderson Maria C. H. , Shaio Cary , Lau Elaine , Dymarskaia Inna , Martin Brian M. , Stubblefield Barbara , Falls Kathleen M. , Carulli John P. , Keith Tim P. , Fann Cathy S. J. , Lacy Lucy G. , Allen Cleona R. , Hostetter Abram M. , Elston Robert C. , Schork Nicholas J. , Egeland Janice A. , Paul Steven M.
Published: July 2002

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at
D4S2949
(maximum
genehunter-plus
nonparametric linkage score = 4.05,
P
= 5.22 × 10
−4
;
sibpal
P
empirical
value <3 × 10
−5
) and suggestive evidence for a locus on chromosome 4q at
D4S397
(maximum
genehunter-plus
nonparametric linkage score = 3.29,
P
= 2.57 × 10
−3
;
sibpal
P
empirical
value <1 × 10
−3
) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.
Host-derived amino acids support the proliferation of symbiotic bacteria
Authors: Graf Joerg , Ruby Edward G.
Published: July 2002

Animals are typically colonized by diverse bacterial symbionts, many of which are commensal and, in numerous cases, even essential for their host’s proper development and growth. In exchange, the host must supply a sufficient array and quantity of nutrients to support the proliferation and persistence of its microbial community. In this investigation, we have examined such a nutritional environment by determining the symbiotic competence of auxotrophic mutants of the bioluminescent bacteriumVibrio fischeri, and have demonstrated that the host squidEuprymna scolopesprovides at least 9 aa to the growing culture of symbioticV. fischeripresent in its light-emitting organ. We also collected and analyzed the extracellular fluid from this organ, in which the symbionts reside, and confirmed that it contained significant amounts of amino acids. The combined results suggested that host-derived free amino acids, as well as peptides or proteins, are a source of the amino acids that support the growth of the symbionts. This work describes a technique to sample the symbionts and their surrounding environment without contamination by host tissue components and, in combination with molecular genetic studies, allows the characterization of the nutritional conditions that support a cooperative animal–bacterial symbiosis.
T cell epitopes of insulin defined in HLA-DR4 transgenic mice are derived from preproinsulin and proinsulin
Authors: Congia Mauro , Patel Salil , Cope Andrew P. , De Virgiliis Stefano , Sønderstrup Grete
Published: July 2002

Approximately one-half of Caucasians with newly diagnosed insulin-dependent diabetes mellitus (IDDM) have autoantibodies to insulin, and the majority of those express the HLA-DR4 genotype [Ziegler, R., Alper, C. A., Awdeh, Z. L., Castano, L., Brink, S. J., Soeldner, J. S., Jackson, R. A. & Eisenbarth, G. S. (1991)
Diabetes
40, 709–714]. However, it has been difficult to demonstrate T cell proliferative responses to human insulin in IDDM patients [Durinovic-Bello, I., Hummel, M. & Ziegler, A. G. (1996)
Diabetes
45, 795–800]. We have immunized transgenic mice expressing the susceptible HLA-DR (α1*0101,β1*0401) (hereafter called DRB1*0401) and human CD4 molecules on a murine major histocompatibility complex class II null background, with human preproinsulin (PPI), proinsulin (PI), and insulin and derived large panels of T cell hybridomas to determine the immunogenic epitopes of these proteins. These results show that the prohormones PI or PPI carry the major immunogenic T cell epitope in the DRB1*0401 transgenic mice. The PPI/PI immunodominant epitope LALEGSLQK was localized at the C-peptide/A-chain junction. This T cell epitope PPI/PI LALEGSLQK is unusual because, normally, it is proteolytically destroyed during the maturation of the insulin molecule. Additionally, this T cell epitope is both processed and presented by human DRB1*0401-positive Epstein–Barr virus transformed B cells, and it can also stimulate T cells from the peripheral blood of HLA-DR4-positive patients with type 1 diabetes. These findings may partly explain why susceptibility to type 1 diabetes is associated with HLA-DR4-positive individuals and why T cell responses to the mature insulin protein are rarely detected in IDDM patients.
Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array
Authors: Zakharov S. D. , Lindeberg M. , Griko Y. , Salamon Z. , Tollin G. , Prendergast F. G. , Cramer W. A.
Published: July 2002

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the α-helical content increases from 60–64% to 80–90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 Å
2
on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled α-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.
NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains
Authors: Armand Philippe , Kirshenbaum Kent , Goldsmith Richard A. , Farr-Jones Shauna , Barron Annelise E. , Truong Kiet T. V. , Dill Ken A. , Mierke Dale F. , Cohen Fred E. , Zuckermann Ronald N. , Bradley Erin K.
Published: July 2002

Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five
para
-substituted (
S
)-
N
-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all
cis
-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with
cis
-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.
Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: Implications for cellular iron homeostasis
Authors: Salter-Cid Luisa , Brunmark Anders , Li Yuanhao , Leturcq Didier , Peterson Per A. , Jackson Michael R. , Yang Young
Published: July 2002

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.
Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn
2+
or Cd
2+
Authors: Paddock M. L. , Graige M. S. , Feher G. , Okamura M. Y.
Published: July 2002

The reaction center (RC) from
Rhodobacter sphaeroides
converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule Q
B
(the secondary quinone acceptor). A unique pathway for proton transfer to the Q
B
site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer
k
AB
(2)
(Q
A
−•
Q
B
−•
+ H
+
→ Q
A
(Q
B
H)
−
, where Q
A
is the primary quinone acceptor). Zn
2+
and Cd
2+
bound stoichiometrically to the RC (
K
D
≤ 0.5 μM) and reduced the observed value of
k
AB
(2)
10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the
k
AB
(2)
reaction. In native RCs,
k
AB
(2)
was previously shown to be rate-limited by electron transfer based on the dependence of
k
AB
(2)
on the driving force for electron transfer. Upon addition of Zn
2+
or Cd
2+
,
k
AB
(2)
became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 10
2
-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D
+•
Q
A
Q
B
−•
→ DQ
A
Q
B
suggests that the binding site is located far (>10 Å) from Q
B
. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to Q
B
−•
occurs.
Hsl7p, a negative regulator of Ste20p protein kinase in the
Saccharomyces cerevisiae
filamentous growth-signaling pathway
Authors: Fujita Atsushi , Tonouchi Akio , Hiroko Takatoshi , Inose Fumika , Nagashima Takeyuki , Satoh Rika , Tanaka Shigeko
Published: July 2002

In the budding yeast,
Saccharomyces cerevisiae
, protein kinases Ste20p (p21
Cdc42p/Rac
-activated kinase), Ste11p [mitogen-activated protein kinase (MAPK) kinase kinase], Ste7p (MAPK kinase), Fus3p, and Kss1p (MAPKs) are utilized for haploid mating, invasive growth, and diploid filamentous growth. Members of the highly conserved Ste20p/p65
PAK
protein kinase family regulate MAPK signal transduction pathways from yeast to man. We describe here a potent negative regulator of Ste20p in the yeast filamentous growth-signaling pathway. We identified a mutant,
hsl7
, that exhibits filamentous growth on rich medium. Hsl7p belongs to a highly conserved protein family in eukaryotes. Hsl7p associates with the noncatalytic region within the amino-terminal half of Ste20p as well as Cdc42p. Deletions of
HSL7
in haploid and diploid strains led to cell elongation and enhancement of both haploid invasive growth and diploid pseudohyphal growth. However, deletions of
STE20
in haploid and diploid greatly diminished these
hsl7
-associated phenotypes. In addition, overexpression of
HSL7
inhibited pseudohyphal growth. Thus, Hsl7p may inhibit the activity of Ste20p in the
S. cerevisiae
filamentous growth-signaling pathway. Our genetic analyses suggest the possibility that Cdc42p and Hsl7p compete for binding to Ste20p for pseudohyphal development when starved for nitrogen.
Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1
Authors: Zhang Pu , Behre Gerhard , Pan Jing , Iwama Atsushi , Wara-aswapati Nawarat , Radomska Hanna S. , Auron Philip E. , Tenen Daniel G. , Sun Zijie
Published: July 2002

The process through which multipotential hematopoietic cells commit to distinct lineages involves the induction of specific transcription factors. PU.1 (also known as Spi-1) and GATA-1 are transcription factors essential for the development of myeloid and erythroid lineages, respectively. Overexpression of PU.1 and GATA-1 can block differentiation in lineages in which they normally are down-regulated, indicating that not only positive but negative regulation of these factors plays a role in normal hematopoietic lineage development. Here we demonstrate that a region of the PU.1 Ets domain (the winged helix–turn–helix wing) interacts with the conserved carboxyl-terminal zinc finger of GATA-1 and GATA-2 and that GATA proteins inhibit PU.1 transactivation of critical myeloid target genes. We demonstrate further that GATA inhibits binding of PU.1 to c-Jun, a critical coactivator of PU.1 transactivation of myeloid promoters. Finally, PU.1 protein can inhibit both GATA-1 and GATA-2 transactivation function. Our results suggest that interactions between PU.1 and GATA proteins play a critical role in the decision of stem cells to commit to erythroid vs. myeloid lineages.
How can we boost IQs of “dull children”?: A late adoption study
Authors: Duyme Michel , Dumaret Annick-Camille , Tomkiewicz Stanislaw
Published: July 2002

From 5,003 files of adopted children, 65 deprived children, defined as abused and/or neglected during infancy, were strictly selected with particular reference to two criteria: (
i
) They were adopted between 4 and 6 years of age, and (
ii
) they had an IQ <86 (mean = 77, SD = 6.3) before adoption. The average IQs of adopted children in lower and higher socioeconomic status (SES) families were 85 (SD = 17) and 98 (SD = 14.6), respectively, at adolescence (mean age = 13.5 years). The results show (
i
) a significant gain in IQ dependent on the SES of the adoptive families (mean = 7.7 and mean = 19.5 IQ points in low and high SES, respectively), (
ii
) IQs after adoption are significantly correlated with IQs before adoption, and (
iii
) during adolescence, verbal IQs are significantly lower than performance IQs.
The β-globin promoter is important for recruitment of erythroid Krüppel-like factor to the locus control region in erythroid cells
Authors: Lee Jong-Soo , Lee Chang-Hun , Chung Jay H.
Published: July 2002

Erythroid Krüppel-like factor (EKLF), which binds to the CACCC box in the β-globin promoter, is required for the expression of the β-globin gene in adult erythroid cells. It was recently demonstrated that EKLF is also required for the activity of the β-globin locus control region (LCR) 5′HS3. Some evidence suggests that the LCR and the β-globin promoter interact in adult erythroid cells, and the network of protein–protein interactions that exists between these two elements may regulate how EKLF is recruited to the LCR. In this report, we use the PIN*POINT assay to study the role of the promoter on the recruitment of EKLF to 5′HS2 and 5′HS3 of the LCR. We find that recruitment of EKLF to 5′HS2 requires the TATA box, but recruitment to 5′HS3 depends on the CACCC and TATA boxes of the β-globin promoter. Furthermore, recruitment of EKLF to 5′HS3 only occurred in β-globin-expressing murine erythroid leukemia cells, whereas recruitment of EKLF to 5′HS2 occurred in both γ-globin-expressing K562 cells and murine erythroid leukemia cells. Unlike EKLF, Sp1, which also binds to CACCC boxes, is not recruited to 5′HS3. We have also examined how one 5′HS affects the recruitment of EKLF to another 5′HS. We have found that the recruitment of EKLF to 5′HS3 depends on the presence of 5′HS2 in cis, but the recruitment to 5′HS2 does not depend on 5′HS3. Based on these results, we present a model that illustrates how EKLF may be recruited to the β-globin locus.
In vivo
transcription factor recruitment during thyroid hormone receptor-mediated activation
Authors: Kim Myung K. , Lee Jong-Soo , Chung Jay H.
Published: July 2002

Thyroid hormone receptor (TR) can act as both a transcriptional activator and a silencer. Optimal activation by TR requires synergism with activator(s) bound to the promoter (promoter proximal activator). It is thought that liganded TR either helps to recruit preinitiation complexes (PIC) to the promoter or activates the PIC already recruited. However, the studies analyzing the TR action on the PIC formation were done
in vitro
and, therefore, it is not clear how relevant they are to the
in vivo
TR action. For example,
in vivo
, the TR can act from distances equal to or greater than a kilobase from the promoter, but such distant effect is not reproducible
in vitro
. In this study, we used the PIN*POINT (ProteIN POsition Identification with Nuclease Tail) assay to define the molecular mechanism of TR action on transcription from the thymidine kinase promoter in the cellular context. We demonstrate that the recruitment of promoter-proximal activator Sp1, and the components of the basal transcription factors such as TBP, TFIIB, and Cdk7, is enhanced with thyroid hormone activation. Our results suggest that DNA forms a loop with TR-mediated activation to accommodate interactions between the liganded TR complex and the complex formed on the promoter. We also show that Sp1 bound to the promoter is essential for the DNA looping and recruitment of basal transcription factors such as TFIIB and Cdk7 but not for recruitment of TBP. On the basis of these findings, we present a model that illustrates the molecular mechanism of TR-mediated activation
in vivo
.
Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins
Authors: Whitelegge J. P. , le Coutre J. , Lee J. C. , Engel C. K. , Privé G. G. , Faull K. F. , Kaback H. R.
Published: July 2002

Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane α-helices, the full-length lactose permease from
Escherichia coli
. Furthermore, ESI-MS is used to titrate reactive thiols with
N
-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.
Does canvassing increase voter turnout? A field experiment
Authors: Gerber Alan S. , Green Donald P.
Published: July 2002

This paper reports the results of a randomized field experiment involving registered voters in the city of New Haven. Nonpartisan get-out-the-vote messages were delivered through personal canvassing shortly before the November 1998 election. We find that personal canvassing increased voter turnout by ≈6. The effect of personal contact seems to be slightly smaller for voters registered with a major political party and higher for unaffiliated voters, although the hypothesis that all voters are equally affected could not be rejected. Study of several alternative political messages provided equivocal evidence suggesting the superiority of a canvassing appeal that emphasizes the closeness of the election.
Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice
Authors: Eguchi Naomi , Minami Toshiaki , Shirafuji Naoki , Kanaoka Yoshihide , Tanaka Takashi , Nagata Akihisa , Yoshida Nobuaki , Urade Yoshihiro , Ito Seiji , Hayaishi Osamu
Published: July 2002

Prostaglandin (PG) D
2
is the most abundant prostanoid produced in the central nervous system of mammals and has been implicated in the modulation of neural functions such as sleep induction, nociception, regulation of body temperature, and odor responses. We generated gene-knockout mice for lipocalin-type PGD
2
synthase (L-PGDS) and found that the intrathecal administration of PGE
2
, an endogenous pain-producing substance, failed to elicit allodynia (touch-evoked pain), which is one typical phenomenon of neuropathic pain, whereas it evoked thermal hyperalgesia, in L-PGDS−/− mice. We also found that the allodynic response induced by the γ-aminobutyric acid (GABA)
A
receptor antagonist bicuculline was selectively abolished in the L-PGDS−/− mice, among excitatory and inhibitory agents that induced allodynia in wild-type mice. Interestingly, simultaneous injection of a femtogram amount of PGD
2
with PGE
2
or bicuculline induced allodynia in L-PGDS−/− mice to the same extent as in wild-type mice. The PGE
2
- or bicuculline-evoked allodynia in wild-type and in PGD
2
-supplemented L-PGDS−/− mice was blocked by a PGD
2
receptor antagonist given in a femtogram amount. These results reveal that endogenous PGD
2
is essential for both PGE
2
- and bicuculline-induced allodynia.
A chemically labeled cytotoxic agent: Two-photon fluorophore for optical tracking of cellular pathway in chemotherapy
Authors: Wang Xiaopeng , Krebs Linda J. , Al-Nuri Mohammed , Pudavar Haridas E. , Ghosal Saswati , Liebow Charles , Nagy Attila A. , Schally Andrew V. , Prasad Paras N.
Published: July 2002

Chemotherapy is commonly used in the treatment of cancers. However, the mechanism of action of many of these agents is not well understood. We present the synthesis of a two-photon fluorophore (C625) and its biological application when chemically linked to a chemotherapeutic agent (AN-152). By using two-photon laser-scanning microscopy, the drug:fluorophore conjugate can be observed directly as it interacts with receptor-positive cell lines. The results of this project visually show the receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus. These observations will allow for better understanding of the drug’s therapeutic mechanism, which is a subject of ongoing research aimed at improving present methods for cancer therapy.
Spontaneous and antigen-induced production of HIV-inhibitory β-chemokines are associated with AIDS-free status
Authors: Garzino-Demo Alfredo , Moss Ronald B. , Margolick Joseph B. , Cleghorn Farley , Sill Anne , Blattner William A. , Cocchi Fiorenza , Carlo Dennis J. , DeVico Anthony L. , Gallo Robert C.
Published: July 2002

The β-chemokines RANTES, macrophage inflammatory protein (MIP)-1α, and MIP-1β suppress infection by macrophage-tropic strains of HIV and simian immunodeficiency virus (SIV) by binding and down-regulating the viral coreceptor, CCR5. Accordingly, we have examined whether higher levels of CCR5 ligands are associated with a more favorable clinical status in AIDS. A cross-sectional study of 100 subjects enrolled in the Multicenter AIDS Cohort Study at the Baltimore site was conducted to measure chemokine production and lymphocyte proliferation by peripheral blood mononuclear cells (PBMC). Statistical analyses of the data revealed that the production of HIV-suppressive β-chemokines by HIV antigen-stimulated PBMC was significantly higher in HIV-positive subjects without AIDS compared with subjects with clinical AIDS. Increased chemokine production was also correlated with higher proliferative responses to HIV antigens. Both parameters were significantly lower in the AIDS versus non-AIDS group. Notably, significantly higher levels of MIP-1α were also observed with unstimulated PBMC from seronegative subjects at risk for HIV infection released as compared with seropositive and non-Multicenter AIDS Cohort Study seronegative subjects. The association of chemokine production with antigen-induced proliferative responses, more favorable clinical status in HIV infection, as well as with an uninfected status in subjects at risk for infection suggests a positive role for these molecules in controlling the natural course of HIV infection.
Phylogenetic analysis of “Volvocacae” for comparative genetic studies
Authors: Coleman Annette W.
Published: July 2002

Sequence analysis based on multiple isolates representing essentially all genera and species of the classic family Volvocaeae has clarified their phylogenetic relationships. Cloned internal transcribed spacer sequences (ITS-1 and ITS-2, flanking the 5.8S gene of the nuclear ribosomal gene cistrons) were aligned, guided by ITS transcript secondary structural features, and subjected to parsimony and neighbor joining distance analysis. Results confirm the notion of a single common ancestor, andChlamydomonas reinharditiialone among all sequenced green unicells is most similar. Interbreeding isolates were nearest neighbors on the evolutionary tree in all cases. Some taxa, at whatever level, prove to be clades by sequence comparisons, but others provide striking exceptions. The morphological speciesPandorina morum, known to be widespread and diverse in mating pairs, was found to encompass all of the isolates of the four species ofVolvulina.Platydorinaappears to have originated early and not to fall within the genusEudorina, with which it can sometimes be confused by morphology. The four species ofPleodorinaappear variously associated withEudorinaexamples. Although the species ofVolvoxare each clades, the genusVolvoxis not. The conclusions confirm and extend prior, more limited, studies on nuclear SSU and LSU rDNA genes and plastid-encoded rbcL and atpB. The phylogenetic tree suggests which classical taxonomic characters are most misleading and provides a framework for molecular studies of the cell cycle-related and other alterations that have engendered diversity in both vegetative and sexual colony patterns in this classical family.
Structural origins of the exonuclease resistance of a zwitterionic RNA
Authors: Teplova Marianna , Wallace Scot T. , Tereshko Valentina , Minasov George , Symons Alice M. , Cook P. Dan , Manoharan Muthiah , Egli Martin
Published: July 2002

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-
O
-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the
Escherichia coli
Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-
O
-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.
Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH
Authors: Khaled Annette R. , Kim Kyungjae , Hofmeister Robert , Muegge Kathrin , Durum Scott K.
Published: July 2002

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.
A molecular basis for nitric oxide sensing by soluble guanylate cyclase
Authors: Zhao Yunde , Brandish Philip E. , Ballou David P. , Marletta Michael A.
Published: July 2002

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (
k
on
> 1.4 × 10
8
M
−1
⋅s
−1
at 4°C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (
k
= 2.4 × 10
5
M
−1
⋅s
−1
at 4°C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (
k
= 7.1 ± 2 × 10
8
M
−1
⋅s
−1
at 4°C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.