bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel DNA-binding motif in MarA: The first structure for an AraC family transcriptional activator
Authors: Rhee Sangkee , Martin Robert G. , Rosner Judah L. , Davies David R.
Published: July 2002

A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described. MarA consists of two similar subdomains, each containing a helix–turn–helix DNA-binding motif. The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 Å thereby bending the DNA by ≈35°. Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.
Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts
Authors: Crofts L. A. , Hancock M. S. , Morrison N. A. , Eisman J. A.
Published: July 2002

The effects of 1,25-dihydroxyvitamin D
3
[1,25-(OH)
2
D
3
] are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. We have identified upstream exons of the human (h) VDR gene that are incorporated into variant transcripts, two of which encode N-terminal variant receptor proteins. Expression of the hVDR gene, which spans more than 60 kb and consists of at least 14 exons, is directed by two distinct promoters. A tissue-specific distal promoter generates unique transcripts in tissues involved in calcium regulation by 1,25-(OH)
2
D
3
and can direct the expression of a luciferase reporter gene in a cell line-specific manner. These major N-terminal differences in hVDR transcripts, potentially resulting in structural differences in the expressed receptor, may contribute to cellular responsiveness to 1,25-(OH)
2
D
3
through tissue differences in the regulation of VDR expression.
The
Drosophila
LIM-only gene,
dLMO
, is mutated in
Beadex
alleles and might represent an evolutionarily conserved function in appendage development
Authors: Zeng Chaoyang , Justice Nicholas J. , Abdelilah Salim , Chan Yee-Ming , Jan Lily Y. , Jan Yuh Nung
Published: July 2002

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal–ventral boundary, which functions to direct the development of the
Drosophila
wing. We report that misexpression of dLMO, a
Drosophila
LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal–ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior–posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of
Beadex
, a long-studied dominant mutation that we show disrupts the
dLMO
transcript in the 3′ untranslated region.
dLMO
normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of
dLMO
is expressed in the developing limb bud of the mouse. This indicates that LMO proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages.
In vivo
responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin
Authors: Zeleznik Anthony J.
Published: July 2002

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.
Modulation of cell death by Bcl-x
L
through caspase interaction
Authors: Clem Rollie J. , Cheng Emily H.-Y. , Karp Christopher L. , Kirsch David G. , Ueno Kazuyoshi , Takahashi Atsushi , Kastan Michael B. , Griffin Diane E. , Earnshaw William C. , Veliuona Michael A. , Hardwick J. Marie
Published: July 2002

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-x
L
, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-x
L
. The loop domain of Bcl-x
L
is cleaved by caspases
in vitro
and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-x
L
in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-x
L
, suggesting that interaction of Bcl-x
L
with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-x
L
is cleaved, the C-terminal fragment of Bcl-x
L
potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-x
L
may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-x
L
during the execution phase of cell death converts Bcl-x
L
from a protective to a lethal protein.
Drug concentration heterogeneity facilitates the evolution of drug resistance
Authors: Kepler Thomas B. , Perelson Alan S.
Published: July 2002

Pathogenic microorganisms use Darwinian processes to circumvent attempts at their control through chemotherapy. In the case of HIV-1 infection, in which drug resistance is a continuing problem, we show that in one-compartment systems, there is a relatively narrow window of drug concentrations that allows evolution of resistant variants. When the system is enlarged to two spatially distinct compartments held at different drug concentrations with transport of virus between them, the range of average drug concentrations that allow evolution of resistance is significantly increased. For high average drug concentrations, resistance is very unlikely to arise without spatial heterogeneity. We argue that a quantitative understanding of the role played by heterogeneity in drug levels and pathogen transport is crucial for attempts to control re-emergent infectious disease.
Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-
fos
gene
Authors: De Cesare Dario , Jacquot Sylvie , Hanauer André , Sassone-Corsi Paolo
Published: July 2002

Activation by growth factors of the Ras-dependent signaling cascade results in the induction of p90 ribosomal S6 kinases (p90
rsk
). These are translocated into the nucleus upon phosphorylation by mitogen-activated protein kinases, with which p90
rsk
are physically associated in the cytoplasm. In humans there are three isoforms of the p90
rsk
family, Rsk-1, Rsk-2, and Rsk-3, which are products of distinct genes. Although these isoforms are structurally very similar, little is known about their functional specificity. Recently, mutations in the
Rsk
-2 gene have been associated with the Coffin–Lowry syndrome (CLS). We have studied a fibroblast cell line established from a CLS patient that bears a nonfunctional Rsk-2. Here we document that in CLS fibroblasts there is a drastic attenuation in the induced Ser-133 phosphorylation of transcription factor CREB (cAMP response element-binding protein) in response to epidermal growth factor stimulation. The effect is specific, since response to serum, cAMP, and UV light is unaltered. Furthermore, epidermal growth factor-induced expression of c-
fos
is severely impaired in CLS fibroblasts despite normal phosphorylation of serum response factor and Elk-1. Finally, coexpression of Rsk-2 in transfected cells results in the activation of the c-
fos
promoter via the cAMP-responsive element. Thus, we establish a link in the transduction of a specific growth factor signal to changes in gene expression via the phosphorylation of CREB by Rsk-2.
Retinoic acid X receptor in the diploblast,
Tripedalia
cystophora
Authors: Kostrouch Zdenek , Kostrouchova Marta , Love William , Jannini Emmanuele , Piatigorsky Joram , Rall J. Edward
Published: July 2002

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian,
Tripedalia cystophora
, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-
cis
retinoic acid (
K
d
= 4 × 10
−10
M) and binds to the DNA sequence, PuGGTCA as a monomer
in vitro
. jRXR also heterodimerizes with
Xenopus
TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His
6
)jRXR fusion protein was identified in the promoters of three
T. cystophora
crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate
in vivo
targets for jRXR. Finally, an antibody prepared against (His
6
)jRXR showed that full-length jRXR is expressed at all developmental stages of
T. cystophora
except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.
Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus
Authors: Ramadevi N. , Burroughs Nicholas J. , Mertens Peter P. C. , Jones Ian M. , Roy Polly
Published: July 2002

The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or
in vitro
-produced uncapped BTV RNA transcripts to m
7
GpppGm in the presence of
S
-adenosyl-
l
-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.
AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice
Authors: Nottoli Timothy , Hagopian-Donaldson Stephanie , Zhang Jian , Perkins Archibald , Williams Trevor
Published: July 2002

The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen.
A widely expressed βIII spectrin associated with Golgi and cytoplasmic vesicles
Authors: Stankewich Michael C. , Tse William T. , Peters Luanne L. , Ch’ng Yeang , John Kathryn M. , Stabach Paul R. , Devarajan Prasad , Morrow Jon S. , Lux Samuel E.
Published: July 2002

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the β-spectrin gene family by homology searches of the GenBank databases and by 5′ rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called βIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. βIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human βIII spectrin gene (
SPTBN2
) maps to chromosome 11q13 and the mouse gene (
Spnb3
) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human βIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that βIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and β-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment
in vitro
with βIII spectrin. βIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.
Autoregulation at the level of mRNA 3′ end formation of the
suppressor of forked
gene of
Drosophila melanogaster
is conserved in
Drosophila virilis
Authors: Audibert Agnès , Simonelig Martine
Published: July 2002

The
Drosophila melanogaster
Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in
Drosophila
. The
su(f)
gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive
su(f)
mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated
su(f)
RNA. Cloning of
su(f)
from
Drosophila virilis
and analysis of its RNA profile suggest that
su(f)
autoregulation is conserved in this species. Sequence comparison between
su(f)
from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by
su(f)
. Our results indicate a role of
su(f)
in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.
Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity
Authors: Shotton David M. , Attaran Amir
Published: July 2002

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDbtarget cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDbadhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.
The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway
Authors: Wu Xinyi , Senechal Kristen , Neshat Mehran S. , Whang Young E. , Sawyers Charles L.
Published: July 2002

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.
Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca
2+
channel by ligand tuning of Ca
2+
inhibition
Authors: Mak Don-On Daniel , McBride Sean , Foskett J. Kevin
Published: July 2002

Inositol 1,4,5-tris-phosphate (IP
3
) binding to its receptors (IP
3
R) in the endoplasmic reticulum (ER) activates Ca
2+
release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca
2+
concentration signals including temporal oscillations and propagating waves. IP
3
-mediated Ca
2+
release is also controlled by cytoplasmic Ca
2+
concentration with both positive and negative feedback. Single-channel properties of the IP
3
R in its native ER membrane were investigated by patch clamp electrophysiology of isolated
Xenopus
oocyte nuclei to determine the dependencies of IP
3
R on cytoplasmic Ca
2+
and IP
3
concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca
2+
concentration ([Ca
2+
]
i
) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP
3
, even as [Ca
2+
]
i
was raised to high concentrations, displaying two distinct types of functional Ca
2+
binding sites: activating sites with half-maximal activating [Ca
2+
]
i
(
K
act
) of 210 nM and Hill coefficient (
H
act
) ≈2; and inhibitory sites with half-maximal inhibitory [Ca
2+
]
i
(
K
inh
) of 54 μM and Hill coefficient (
H
inh
) ≈4. Lowering IP
3
concentration was without effect on Ca
2+
activation parameters or
H
inh
, but decreased
K
inh
with a functional half-maximal activating IP
3
concentration (
K

IP
3

) of 50 nM and Hill coefficient (
H

IP
3

) of 4 for IP
3
. These results demonstrate that Ca
2+
is a true receptor agonist, whereas the sole function of IP
3
is to relieve Ca
2+
inhibition of IP
3
R. Allosteric tuning of Ca
2+
inhibition by IP
3
enables the individual IP
3
R Ca
2+
channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca
2+
concentration signaling and quantal Ca
2+
release.
ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin
Authors: Muchowski Paul J. , Clark John I.
Published: July 2002

We report direct experimental evidence that human αB-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS)
in vitro
. In the presence of 3.5 mM ATP, CS reactivation by αB-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of αB-crystallin on the unfolding and aggregation of CS at 45°C. Consistent with these findings, cell viability at 50°C was improved nearly five orders of magnitude in
Escherichia coli
expressing αB-crystallin. SDS/PAGE analysis of cell lysates suggested that αB-crystallin protects cells against physiological stress
in vivo
by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of αB-crystallin as a molecular chaperone both
in vitro
and
in vivo
and describes the enhancement of αB-crystallin chaperone functions by ATP.
Interferon γ-producing γδ T cell-dependent antibody isotype switching in the absence of germinal center formation during virus infection
Authors: Maloy Kevin J. , Odermatt Bernhard , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Ig class switching usually occurs as a consequence of cognate interactions between antigen-specific B cells and CD4+αβ T cells. Vesicular stomatitis virus (VSV) infection of immunocompetent mice induces a rapid T-independent neutralizing IgM response followed by a long-lived T-dependent IgG response. Surprisingly, αβ T cell-deficient (TCRα−/−) mice also produced neutralizing IgG antibodies when infected with live VSV or with a recombinant vaccinia virus expressing the VSV glycoprotein (Vacc-IND-G), but not when immunized with UV-inactivated VSV (UV-VSV). The neutralizing IgG responses did not require the presence of NK cells or complement, but were crucially dependent on IFN-γ and were predominantly of the IgG2a isotype. IgG production depended on residual CD3+non-αβ T cell populations present in the TCRα−/−mice, which produced IFN-γ uponin vitrostimulation. A key role for γδ T cells was confirmed by the fact that TCRβ−/−mice also generated strong neutralizing IgG responses to VSV, whereas TCRβ−/−δ−/−mice produced very low titers. The neutralizing IgG responses of TCRα−/−mice were accompanied by the development of memory B cells, but not by antigen-specific germinal center (GC) formation. Thus, during viral infection of αβ T cell-deficient mice, γδ T cells may provide the signals that are required for isotype switching.
Activation of the atrial K
ACh
channel by the βγ subunits of G proteins or intracellular Na
+
ions depends on the presence of phosphatidylinositol phosphates
Authors: Sui Jin Liang , Petit-Jacques Jérôme , Logothetis Diomedes E.
Published: July 2002

The βγ subunits of GTP-binding proteins (G
βγ
) activate the muscarinic K
+
channel (K
ACh
) in heart by direct binding to both of its component subunits. K
ACh
channels can also be gated by internal Na
+
ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP
2
) mimics the ATP effects and that depletion or block of PIP
2
retards the stimulatory effects of G
βγ
subunits or Na
+
ions on channel activity, effects that can be reversed by restoring PIP
2
. Thus, regulation of K
ACh
channel activity may be crucially dependent on PIP
2
and phosphatidylinositol signaling. These striking functional results are in agreement with
in vitro
biochemical studies on the PIP
2
requirement for G
βγ
stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for G
βγ
-mediated signal transduction.
Vesicles on strings: Morphological evidence for processive transport within the Golgi stack
Authors: Orci Lelio , Perrelet Alain , Rothman James E.
Published: July 2002

Cis
-Golgi cisternae have a higher freeze-fracture particle density than
trans
-cisternae. Transport vesicles neighboring
cis
or
trans
positions of the Golgi stack have a particle concentration comparable to that of the adjacent cisterna and the buds emerging from it. This implies that transport vesicles remain locally within the stack during their lifetime, near their origin, favoring a processive pattern of transport in which vesicle transfers occur preferentially between adjacent cisternae in the stack. A “string theory” is proposed to account for processive transport, in which a carpet of fibrous attachment proteins located at the surface of cisternae (the strings) prevent budded vesicles from diffusing away but still allow them to diffuse laterally, effectively limiting transfers to adjoining cisternae in the stack. Fibrous elements that multivalently connect otherwise free COPI-coated vesicles and uncoated transport vesicles to one or two cisternae simultaneously are discerned readily by electron microscopy. It is suggested that long, coiled coil, motif-rich, Golgi-specific proteins including p115, GM130, and possibly giantin, among others, function as the proposed strings.
Human papillomavirus type 16
E6
and
E7
oncogenes abrogate radiation-induced DNA damage responses
in vivo
through p53-dependent and p53-independent pathways
Authors: Song Shiyu , Gulliver Gene A. , Lambert Paul F.
Published: July 2002

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors
in vivo
. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses
in vivo
. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7’s transforming function, were also required for E7 to modulate DNA damage responses
in vivo
. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.
Unequal representation of cardinal and oblique contours in ferret visual cortex
Authors: Coppola David M. , White Leonard E. , Fitzpatrick David , Purves Dale
Published: July 2002

We have measured the amount of cortical space activated by differently oriented gratings in 25 adult ferrets by optical imaging of intrinsic signal. On average, 7% more area of the exposed visual cortex was preferentially activated by vertical and horizontal contours than by contours at oblique angles. This anisotropy may reflect the real-world prevalence of contours in the cardinal axes and could explain the greater sensitivity of many animals to vertical and horizontal stimuli.
Comparison of the auditory systems of heterosexuals and homosexuals: Click-evoked otoacoustic emissions
Authors: McFadden Dennis , Pasanen Edward G.
Published: July 2002

Click-evoked otoacoustic emissions (CEOAEs) are echo-like waveforms emitted by normal-hearing cochleas in response to a brief transient. CEOAEs are known to be stronger in females than in males. In this experiment, the CEOAEs of homosexual and bisexual females were found to be intermediate to those of heterosexual females and heterosexual males. A parsimonious explanation is that the auditory systems of homosexual and bisexual females, and the brain structures responsible for their sexual orientation, have been partially masculinized by exposure to high levels of androgens prenatally. No difference in CEOAEs was observed between homosexual and heterosexual males.
Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance
Authors: Tikkanen Matti J. , Wähälä Kristiina , Ojala Sirpa , Vihma Veera , Adlercreutz Herman
Published: July 2002

The oxidation of low density lipoproteins (LDLs) is thought to take place in the arterial intima when the particles have become isolated from circulating water-soluble antioxidants. We hypothesized that isoflavonoid antioxidants derived from soy could be incorporated into lipoproteins and possibly could protect them against oxidation, which is regarded as atherogenic. Six healthy volunteers received 3 soy bars [containing the isoflavonoid antioxidants genistein (12 mg) and daidzein (7 mg)] daily for 2 weeks. LDLs were isolated from blood drawn at the the end of a 2-week dietary baseline period, after 2 weeks on soy, and after discontinuation of soy. Large increases in plasma isoflavonoid levels occurred during soy feeding, but only minute amounts were stably associated with lipoproteins (less than 1% of plasma isoflavonoids in the LDL fraction). The LDLs were subjected to copper-mediated oxidation
in vitro
. Compared with off soy values, lag phases of LDL oxidation curves were prolonged by a mean of 20 min (
P
< 0.02) during soy intake, indicating a reduced susceptibility to oxidation. The results suggest that intake of soy-derived antioxidants, such as genistein and daidzein, may provide protection against oxidative modification of LDL. As only very small amounts of these substances were detected in purified LDL, modified LDL particles may have been produced
in vivo
by circulating isoflavonoids promoting resistance to oxidation
ex vivo
.
Differential colocalization of estrogen receptor β (ERβ) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: An immunocytochemical study
Authors: Alves Stephen E. , Lopez Veronica , McEwen Bruce S. , Weiland Nancy G.
Published: July 2002

Evidence exists for the localization of the newly identified estrogen receptor β (ERβ) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERα. Presently, we investigate whether ERβ-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague–Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17β (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERβ antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERβ-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERβ-ir within magnocellular areas was noticeably less intense. OT-/ERβ-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains (≈50% of OT and 25% of ERβ-labeled cells between bregma −1.78 and −2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERβ-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERβ-ir. In the SON, most nuclear ERβ-ir colocalized with AVP-ir, whereas few OT-/ERβ-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERβ-mediated mechanism, in a tissue-specific manner.
Shape and roughness activate different somatosensory areas in the human brain
Authors: Roland Per E. , O’Sullivan Brendan , Kawashima Ryuta
Published: July 2002

Somatosensory stimuli are known to activate the postcentral gyrus,
and neurons there fire when the skin is in contact with objects. Also
neurons in the lateral fissure, the parietal operculum, fire when rough
surfaces are felt. However the localization of somatosensory
association areas in humans is largely unknown and differences in
functional contributions between somatosensory association areas has
not been previously demonstrated. For these reasons the regional
cerebral blood flow was measured with
15
O-butanol and
positron-emission tomography in two groups of young volunteers
discriminating the lengths, shapes, and roughness of objects with their
right hand. Roughness discrimination activated the lateral parietal
opercular cortex significantly more than did length or shape
discrimination. A Boolean intersection of the cluster images showing
the statistical significant increases of length and shape
discrimination demonstrated that shape and length discrimination
activated the same cortical field lining the anterior part of the
intraparietal sulcus (IPA). Shape and length discrimination activated
IPA significantly more than did roughness discriminaton. These findings
demonstrate a separation in functional contributions of lateral
parietal opercular cortex and IPA. The results indicate different
cortical processing streams for the somatosensory submodalities
microgeometry and macrogeometry.
Myeloid-specific transcriptional activation by murine myeloid zinc-finger protein 2
Authors: Murai Kasumi , Murakami Hiroshi , Nagata Shigekazu
Published: July 2002

Myeloid zinc finger protein 2 (MZF-2) is a zinc-finger transcription factor that is expressed in myeloid cells, particularly in the cells committed to the neutrophilic lineage. Here we examine the ability of murine MZF-2 (mMZF-2) to activate transcription. The mMZF-2 protein binds to a DNA element (MZF-binding site) through its zinc-finger domain. When the intact mMZF-2 was cotransfected with a reporter gene, it did not activate transcription. However, N-terminal deletion mutants greatly enhanced transcription specifically in myeloid cells. Furthermore, in an
in vivo
competition assay, the middle region of MZF-2 inhibited the mMZF-2-mediated transcription activation. These results suggest that mMZF-2 is a transcriptional factor that can specifically work in myeloid cells and can be divided into at least three functional domains. The N-terminal domain inhibits transactivation by masking the effect of the activation domain. The middle region recruits a coactivator, which is responsible for myeloid-specific transcriptional activation. The C-terminal zinc-finger domain functions as a DNA-binding domain.
Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: Evidence for multiple critical loci
Authors: Liang Hong , Fairman Jeff , Claxton David F. , Nowell Peter C. , Green Eric D. , Nagarajan Lalitha
Published: July 2002

Complete or partial deletions of the long arm of chromosome 7 (7q- and -7) are nonrandom abnormalities seen in primary and therapy-induced myelodysplasia (MDS) and acute myelogenous leukemia (AML). Monosomy 7, occurring as the sole cytogenetic anomaly in a small but significant number of cases, may denote a dominant mechanism involving critical tumor suppressor gene(s). We have determined the extent of allele loss in cytogenetically prescreened MDS and AML patients for microsatellite markers from chromosome 7q22 and 7q31. Whereas >80% of these cases revealed allele loss for the entire region, a rare case of the 7q- chromosome showed allele loss for only the proximal 7q31.1 loci flanked by the markers
D7S486
and
D7S2456
, and a case of monosomy 7 revealed allele loss for loci at both 7q31 and 7q22 with retention of sequences between these sets of loci. Furthermore, a case of AML with no cytogenetic anomaly of chromosome 7 revealed a submicroscopic allelic imbalance for a third distal locus,
D7S677
. These findings suggest the presence of three distinct critical loci that may contribute alone or in combination to the evolution of MDS and AML. The data also provide molecular evidence for unbalanced translocation with noncontiguous deletions, as an alternate mechanism underlying monosomy 7.
Interferon α and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS
Authors: Zagury Daniel , Lachgar Abderrahim , Chams Vida , Fall Lat S. , Bernard Jacky , Zagury Jean-François , Bizzini Bernard , Gringeri Alessandro , Santagostino Elena , Rappaport Jay , Feldman Michael , Burny Arsène , Gallo Robert C.
Published: July 2002

HIV type 1 (HIV-1) not only directly kills infected CD4+T cells but also induces immunosuppression of uninfected T cells. Two immunosuppressive proteins, interferon α (IFNα) and extracellular Tat, mediate this process because specific antibodies against these proteins prevent generation of suppressor cells in HIV-1-infected peripheral blood mononuclear cell cultures. Furthermore, the production of C-C chemokines in response to immune cell activation, initially enhanced by IFNα and Tat, ultimately is inhibited by these proteins in parallel with their induction of immunosuppression. The clinical corollary is the immunosuppression of uninfected T cells and the decline in C-C chemokine release found at advanced stages of HIV-1 infection paralleling rising levels of IFNα and extracellular Tat. We, therefore, suggest that IFNα and Tat may be critical targets for anti-AIDS strategies.
The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex
Authors: Rozenblatt–Rosen Orit , Rozovskaia Tanya , Burakov Darya , Sedkov Yurii , Tillib Sergei , Blechman Janna , Nakamura Tatsuya , Croce Carlo M. , Mazo Alexander , Canaani Eli
Published: July 2002

The
ALL-1
gene was discovered by virtue of its involvement in human acute leukemia. Its
Drosophila
homolog
trithorax
(
trx
) is a member of the
trx-Polycomb
gene family, which maintains correct spatial expression of the
Antennapedia
and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of
Drosophila
cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of
snr
1, which is classified as a
trx
group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by
in vitro
binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/
trx
targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.
DnaA-stimulated transcriptional activation of
ori
λ:
Escherichia coli
RNA polymerase β subunit as a transcriptional activator contact site
Authors: Szalewska-Palasz Agnieszka , Wegrzyn Alicja , Blaszczak Adam , Taylor Karol , Wegrzyn Grzegorz
Published: July 2002

We present evidence that
Escherichia coli
RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the
p
R
promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the
p
R
promoter
in vitro
. Particular mutations in the
rpoB
gene were able to suppress negative effects that certain
dnaA
mutations had on the replication of λ plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the
p
R
promoter was scrambled by
in vitro
mutagenesis, the
p
R
promoter was no longer activated by DnaA both
in vivo
and
in vitro
. Therefore, we conclude that DnaA may contact the β subunit of RNA polymerase during activation of the
p
R
promoter. A new classification of prokaryotic transcriptional activators is proposed.
The lethal mutation of the mouse wasted (
wst
) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1α, encoded by the
Eef1a2
gene
Authors: Chambers Doreen M. , Peters Josephine , Abbott Catherine M.
Published: July 2002

We have identified the mutation responsible for the autosomal recessive wasted (
wst
) mutation of the mouse. Wasted mice are characterized by wasting and neurological and immunological abnormalities starting at 21 days after birth; they die by 28 days. A deletion of 15.8 kb in wasted mice abolishes expression of a gene called
Eef1a2
, encoding a protein that is 92% identical at the amino acid level to the translation elongation factor EF1α (locus
Eef1a
). We have found no evidence for the involvement of another gene in this deletion. Expression of
Eef1a2
is reciprocal with that of
Eef1a
. Expression of
Eef1a2
takes over from
Eef1a
in heart and muscle at precisely the time at which the wasted phenotype becomes manifest. These data suggest that there are tissue-specific forms of the translation elongation apparatus essential for postnatal survival in the mouse.
Diploid hybrid speciation inPenstemon(Scrophulariaceae)
Authors: Wolfe Andrea D. , Xiang Qiu-Yun , Kephart Susan R.
Published: July 2002

Hybrid speciation has played a significant role in the evolution of angiosperms at the polyploid level. However, relatively little is known about the importance of hybrid speciation at the diploid level. Two species ofPenstemonhave been proposed as diploid hybrid derivatives based on morphological data, artificial crossing studies, and pollinator behavior observations:Penstemon spectabilis(derived from hybridization betweenPenstemon centranthifoliusandPenstemon grinnellii) andPenstemon clevelandii(derived from hybridization betweenP. centranthifoliusandP. spectabilis). Previous studies were inconclusive regarding the purported hybrid nature of these species because of a lack of molecular markers sufficient to differentiate the parental taxa in the hybrid complex. We developed hypervariable nuclear markers using inter-simple sequence repeat banding patterns to test these classic hypotheses of diploid hybrid speciation inPenstemon. Each species in the hybrid complex was genetically distinct, separated by 10–42 species-specific inter-simple sequence repeat markers. Our data do not support the hybrid origin ofP. spectabilisbut clearly support the diploid hybrid origin ofP. clevelandii. Our results further suggest that the primary reason diploid hybrid speciation is so difficult to detect is the lack of molecular markers able to differentiate parental taxa from one another, particularly with recently diverged species.
A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins
in vivo
Authors: Stagljar Igor , Korostensky Chantal , Johnsson Nils , te Heesen Stephan
Published: July 2002

A detection system for interactions between membrane proteins
in vivo
is described. The system is based on split-ubiquitin [Johnsson, N. & Varshavsky, A. (1994)
Proc. Natl. Acad. Sci. USA
91, 10340–10344]. Interaction between two membrane proteins is detected by proteolytic cleavage of a protein fusion. The cleavage releases a transcription factor, which activates reporter genes in the nucleus. As a result, interaction between membrane proteins can be analyzed by the means of a colorimetric assay. We use membrane proteins of the endoplasmic reticulum as a model system. Wbp1p and Ost1p are both subunits of the oligosaccharyl transferase membrane protein complex. The Alg5 protein also localizes to the membrane of the endoplasmic reticulum, but does not interact with the oligosaccharyltransferase. Specific interactions are detected between Wbp1p and Ost1p, but not between Wbp1p and Alg5p. The new system might be useful as a genetic and biochemical tool for the analysis of interactions between membrane proteins
in vivo
.
Thermodynamic basis of the enhanced specificity of structured DNA probes
Authors: Bonnet Grégoire , Tyagi Sanjay , Libchaber Albert , Kramer Fred Russell
Published: July 2002

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.