bluuebunny/crossref_metadata_2025 huggingface dataset:
Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome
Authors: Preisig-Müller Regina , Schlichthörl Günter , Goerge Tobias , Heinen Steffen , Brüggemann Andrea , Rajan Sindhu , Derst Christian , Veh Rüdiger W. , Daut Jürgen
Published: July 2002

Andersen's syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersen's syndrome. The following results show that Kir2.x channels can form functional heteromers: (
i
) HEK293 cells transfected with Kir2.x–Kir2.y concatemers expressed inwardly rectifying K
+
channels with a conductance of 28–30 pS. (
ii
) Expression of Kir2.x–Kir2.y concatemers in
Xenopus
oocytes produced inwardly rectifying, Ba
2+
sensitive currents. (
iii
) When Kir2.1 and Kir2.2 channels were coexpressed in
Xenopus
oocytes the IC
50
for Ba
2+
block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (
iv
) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (
v
) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (
vi
) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersen's syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersen's syndrome.
Molecular cloning and characterization of a lipid phosphohydrolase that degrades sphingosine-1- phosphate and induces cell death
Authors: Mandala Suzanne M. , Thornton Rosemary , Galve-Roperh Ismael , Poulton Samantha , Peterson Courtney , Olivera Ana , Bergstrom James , Kurtz Myra B. , Spiegel Sarah
Published: July 2002

Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with
LBP1
, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, we report here the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP1 can partially substitute for the function of
LBP1
. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate. Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, our results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate.
Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated
Escherichia coli
need not involve recombination
Authors: Courcelle Justin , Hanawalt Philip C.
Published: July 2002

Alternative reproductive cycles make use of different strategies to generate different reproductive products. In
Escherichia coli
,
recA
and several other
rec
genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires
uvrA
and
uvrC
, as well as
recA
,
recF
, and
recR
. The
rec
genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the
recJ
and
recQ
genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several
rec
gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the
rec
genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.
Intestinal adenomas can develop with a stable karyotype and stable microsatellites
Authors: Haigis Kevin M. , Caya James G. , Reichelderfer Mark , Dove William F.
Published: July 2002

Loss of function of the adenomatous polyposis coli (
APC
)/
Apc
tumor suppressor gene occurs early in the etiology of intestinal cancer in mammals. In human colonic tumors, genomic instability is proposed to be associated with tumor initiation by inducing loss of
APC
function. We have used a mouse model of inherited intestinal cancer (
Apc

Min

/+,
Min
/+) to analyze the earliest stages of tumorigenesis in this organ. We find that tumors from C57BL/6
Min
/+ mice have a stable karyotype and stable microsatellites. In contrast to previous claims, we find that homozygosity for the
Min
allele of
Apc
in tumors can proceed by homologous somatic recombination. Further, our analysis of early, benign human colorectal adenomas failed to reveal any evidence for generalized chromosomal or microsatellite instability. These results cast doubt on the hypothesis that either of these forms of genomic instability is necessary for the initial development of colorectal adenomas. We contrast our analysis of autochthonous primary tumors to other studies involving xenografts or cultured cells.
Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution
Authors: Silva R. A. G. D. , Kubelka Jan , Bour Petr , Decatur Sean M. , Keiderling Timothy A.
Published: July 2002

Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C.
Ab initio
level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.
Aromatase (
Cyp19
) expression is up-regulated by targeted disruption of
Dax1
Authors: Wang Zhen J. , Jeffs Baxter , Ito Masafumi , Achermann John C. , Yu Richard N. , Hales Dale B. , Jameson J. Larry
Published: July 2002

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human
DAX1
gene (also known as
AHC
) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of
Dax1
-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of
Dax1
disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from
Dax1
-deficient male mice. Expression of the aromatase (
Cyp19
) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g.,
StAR
and
Cyp11a
) was not altered. In
in vitro
transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the
Cyp19
promoter but did not inhibit the
StAR
or
Cyp11a
promoters. Elevated
Cyp19
expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the
Dax1
-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these
in vivo
and
in vitro
analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased
Cyp19
expression may account, in part, for the infertility and LCH in
Dax1
-deficient mice.
A systematic exploration of the influence of the protein stability on amyloid fibril formation
in vitro
Authors: Ramírez-Alvarado Marina , Merkel Jane S. , Regan Lynne
Published: July 2002

There are a number of diseases in which normally soluble proteins associate into regular, insoluble amyloid fibrils. The development of
in vitro
model systems in which detailed structural, kinetic, and thermodynamic characterization are feasible is of critical importance to our understanding of the amyloid fibril phenomenon. The formation of amyloid fibrils by proteins that are not associated with disease has been recently described, suggesting that this may be a common property of many proteins and not only of the few proteins associated with amyloidoses. The B1 Ig-binding domain of protein G (β1) is an extremely well-characterized model system. We have found that under certain experimental conditions, some variants of β1 form fibrils with high reproducibility. By controlling the stability of the protein—either by mutations or by changing experimental conditions—we are able to modulate the ability of the protein to form fibrils. For all of the variants, we find that the key requirement for fibril formation is to choose conditions in which the population of intermediate conformations present during the unfolding transition is maximized.
A Δ9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in
Drosophila melanogaster
Authors: Dallerac Renaud , Labeur Carole , Jallon Jean-Marc , Knipple Douglas C. , Roelofs Wendell L. , Wicker-Thomas Claude
Published: July 2002

Drosophila melanogaster
cuticular pheromones consist of unsaturated hydrocarbons with at least one double bond in position 7: 7 tricosene (T) in males and 7,11 heptacosadiene (HD) in females. However, in many African populations like the Tai strain, females possess low levels of 7,11 HD and high levels of its positional isomer 5,9 HD. We have previously isolated a desaturase gene,
desat1
, from the Canton-S strain (CS), a 7,11 HD-2-rich morph of
D. melanogaste
r. This desaturase is located in 87C, a locus that has been involved in the difference between 7,11 HD and 5,9 HD morphs. Therefore, we have searched for different desaturase isoforms in both strains. We first cloned
desat1
in the Tai strain and report here functional expression of
desat1
in CS and Tai. In both strains, the Desat1 enzymes have the same Δ9 specificity and preferentially use palmitate as a substrate, leading to the synthesis of ω7 fatty acids. Also found was a desaturase sequence, named
desat2
, with a homologous catalytic domain and a markedly different N-terminal domain compared with
desat1
. In CS genome, it lies 3.8 kb upstream of
desat1
and is not transcribed in either sex. In the Tai strain, it is expressed only in females and acts preferentially on myristate, leading to the synthesis of ω5 fatty acids. We suggest, therefore, that
desat2
might play a control role in the biosynthesis of 5,9 HD hydrocarbons in Tai females and could explain the dienic hydrocarbon polymorphism in
D. melanogaster.
DNA damage-inducible gene
p33ING2
negatively regulates cell proliferation through acetylation of p53
Authors: Nagashima Makoto , Shiseki Masayuki , Miura Koh , Hagiwara Koichi , Linke Steven P. , Pedeux Remy , Wang Xin W. , Yokota Jun , Riabowol Karl , Harris Curtis C.
Published: July 2002

The p33ING1 protein is a regulator of cell cycle, senescence, and
apoptosis. Three alternatively spliced transcripts of
p33ING1
encode p47ING1a, p33ING1b, and p24ING1c. We
cloned an additional ING family member,
p33ING2
/
ING1L
. Unlike p33ING1b, p33ING2
is induced by the DNA-damaging agents etoposide and
neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell
growth and survival in a p53-dependent manner through induction of
G
1
-phase cell-cycle arrest and apoptosis. p33ING2
strongly enhances the transcriptional-transactivation activity of p53.
Furthermore, p33ING2 expression increases the acetylation of p53 at
Lys-382. Taken together,
p33ING2
is a DNA
damage-inducible gene that negatively regulates cell proliferation
through activation of p53 by enhancing its acetylation.
Growth hormone interacts with the Marek's disease virus SORF2 protein and is associated with disease resistance in chicken
Authors: Liu Hsiao-Ching , Kung Hsing-Jien , Fulton Janet E. , Morgan Robin W. , Cheng Hans H.
Published: July 2002

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by a herpesvirus, the MD virus (MDV). Because MD is a significant economic problem to the poultry industry, there is great interest in enhancing genetic resistance, which is controlled by multiple genes. The influence of the MHC has been clearly demonstrated, and several relevant quantitative trait loci have been mapped; however, no single gene influencing MD resistance has been identified. Transcription ofSORF2is perturbed in the MDV recombinant clone RM1 due to a solo insertion of the reticuloendotheliosis virus long terminal repeat, which may explain the loss of oncogenicity for this strain. Hypothesizing that SORF2-interacting host proteins are involved in MD resistance, we screened a chicken splenic cDNA library by the yeast two-hybrid assay using SORF2 as bait. The chicken growth hormone (GH) structural peptide was identified, and the specific interaction was verified by coimmunoprecipitation. Immunohistochemical staining and indirect immunofluorescence assay indicated that GH and SORF2 can be coexpressed in MDV-infected cells bothin vitroandin vivo. Furthermore, polymorphism in the GH gene (GH1) is associated with the number of tissues with tumors in commercial White Leghorn chickens with the MHCB*2/B*15genotype. We conclude thatGH1may well be a MD resistance gene.
Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7
Authors: Hollis Thomas , Stattel James M. , Walther Dane S. , Richardson Charles C. , Ellenberger Tom
Published: July 2002

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a
single-stranded DNA (ssDNA) binding protein that has essential roles in
DNA replication and recombination. In addition to binding DNA, gp2.5
physically interacts with T7 DNA polymerase and T7 primase-helicase
during replication to coordinate events at the replication fork. We
have determined a 1.9-Å crystal structure of gp2.5 and show that it
has a conserved OB-fold (oligosaccharide/oligonucleotide binding
fold) that is well adapted for interactions with ssDNA. Superposition
of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a
conserved patch of aromatic residues that stack against the bases of
ssDNA in the other crystal structures, suggesting that gp2.5 binds to
ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5
protein, which is required for dimer formation and for interactions
with the T7 DNA polymerase and the primase-helicase, appears to be
flexible and may act as a switch that modulates the DNA binding
affinity of gp2.5.
Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the
Pkd1
gene
Authors: Boulter Catherine , Mulroy Sharon , Webb Sandra , Fleming Stewart , Brindle Kevin , Sandford Richard
Published: July 2002

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation in the kidney, liver, and pancreas and is associated often with cardiovascular abnormalities such as hypertension, mitral valve prolapse, and intracranial aneurysms. It is caused by mutations in
PKD1
or
PKD2
, encoding polycystin-1 and -2, which together form a cell surface nonselective cation ion channel.
Pkd2
−/− mice have cysts in the kidney and pancreas and defects in cardiac septation, whereas

Pkd1
del34

−/− and

Pkd1
L

−/− mice have cysts but no cardiac abnormalities, although vascular fragility was reported in the latter. Here we describe mice carrying a targeted mutation in
Pkd1
(

Pkd1
del17–21βgeo

), which defines its expression pattern by using a
lacZ
reporter gene and may identify novel functions for polycystin-1. Although

Pkd1
del17–21βgeo

+/− adult mice develop renal and hepatic cysts,

Pkd1
del17–21βgeo

−/− embryos die at embryonic days 13.5–14.5 from a primary cardiovascular defect that includes double outflow right ventricle, disorganized myocardium, and abnormal atrio-ventricular septation. Skeletal development is also severely compromised. These abnormalities correlate with the major sites of
Pkd1
expression. During nephrogenesis,
Pkd1
is expressed in maturing tubular epithelial cells from embryonic day 15.5. This expression coincides with the onset of cyst formation in

Pkd1
del34

−/−,

Pkd1
L

−/−, and
Pkd2
−/− mice, supporting the hypothesis that polycystin-1 and polycystin-2 interact
in vivo
and that their failure to do so leads to abnormalities in tubule morphology and function.
Extraocular muscle is defined by a fundamentally distinct gene expression profile
Authors: Porter J. D. , Khanna S. , Kaminski H. J. , Rao J. S. , Merriam A. P. , Richmonds C. R. , Leahy P. , Li J. , Andrade F. H.
Published: July 2002

Skeletal muscle fibers are defined by patterned covariation of key traits that determine contractile and metabolic characteristics. Although the functional properties of most skeletal muscles result from their proportional content of a few conserved muscle fiber types, some, typically craniofacial, muscles exhibit fiber types that appear to lie outside the common phenotypic range. We analyzed gene expression profiles of three putative muscle classes, limb, masticatory, and extraocular muscle (EOM), in adult mice by high-density oligonucleotide arrays. Pairwise comparisons using conservative acceptance criteria identified expression differences in 287 genes between EOM and limb and/or masticatory muscles. Use of significance analysis of microarrays methodology identified up to 400 genes as having an EOM-specific expression pattern. Genes differentially expressed in EOM reflect key aspects of muscle biology, including transcriptional regulation, sarcomeric organization, excitation-contraction coupling, intermediary metabolism, and immune response. These patterned differences in gene expression define EOM as a distinct muscle class and may explain the unique response of these muscles in neuromuscular diseases.
Phosphorylation of UBF at serine 388 is required for interaction with RNA polymerase I and activation of rDNA transcription
Authors: Voit Renate , Grummt Ingrid
Published: July 2002

Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G
1
-specific cyclin-dependent kinase (cdk)/cyclin complexes. After G
1
progression UBF is phosphorylated at serine 388 by cdk2/cyclin E and cdk2/cyclin A. Conversion of serine 388 to glycine abolishes UBF activity, whereas substitution by aspartate enhances the transactivating function of UBF. Protein–protein interaction studies reveal that phosphorylation at serine 388 is required for the interaction between RNA polymerase I and UBF. The results suggest that phosphorylation of UBF represents a powerful means of modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent fashion.
A phylogenetic approach to following West Nile virus in Connecticut
Authors: Anderson John F. , Vossbrinck Charles R. , Andreadis Theodore G. , Iton Anthony , Beckwith William H. , Mayo Donald R.
Published: July 2002

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America.
Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse
Authors: Fujiwara Takeshi , Dunn N. Ray , Hogan Brigid L. M.
Published: July 2002

Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Rather, previous genetic analysis in the mouse has shown that bone morphogenetic protein 4 (Bmp4) is required for the formation of both PGCs and allantois. Bmp4 is expressed in both the trophoblast-derived extraembryonic ectoderm (ExE) and in the epiblast-derived extraembryonic mesoderm (ExM), in which the PGCs, allantois primordium, and angioblasts are first detected. We have shown that Bmp4 made in the ExE functions to induce precursors of PGCs and allantois in the adjacent epiblast, resulting in complete lack of both cell types in homozygous null mutants. However, the function of Bmp4 in the ExM is totally unknown. To address this question, we generated tetraploid (4N) chimeras by aggregating
Bmp4
null ES cells with wild-type tetraploid embryos. In this combination, wild-type tetraploid cells contribute to the extraembryonic trophoblast and primitive endoderm lineages but are excluded from the epiblast and its derivatives, including the ExM. Our results clearly demonstrate that Bmp4 made in the ExM does not affect the establishment of either PGC or allantois lineages, but is required for PGC localization and survival and for the differentiation of the allantois. These findings suggest that
Bmp4
expressed in epiblast-derived tissues plays vital roles in reproduction by regulating both the development of the germ line and the vascular connection between the embryo and the placenta.
The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum
Authors: Fang Shengyun , Ferrone Marco , Yang Cuihong , Jensen Jane P. , Tiwari Swati , Weissman Allan M.
Published: July 2002

gp78, also known as the tumor autocrine motility factor
receptor, is a transmembrane protein whose expression is
correlated with tumor metastasis. We establish that gp78 is a RING
finger-dependent ubiquitin protein ligase (E3) of the endoplasmic
reticulum (ER). Consistent with this, gp78 specifically recruits
MmUBC7, a ubiquitin-conjugating enzyme (E2) implicated in ER-associated
degradation (ERAD), through a region distinct from the RING finger.
gp78 can target itself for proteasomal degradation in a RING finger-
and MmUBC7-dependent manner. Importantly, gp78 can also mediate
degradation of CD3-δ, a well-characterized ERAD substrate. In
contrast, gp78 lacking an intact RING finger or its multiple
membrane-spanning domains stabilizes CD3-δ. gp78 has thus been
found to be an example of a mammalian cellular E3 intrinsic to
the ER, suggesting a potential link between ubiquitylation,
ERAD, and metastasis.
A new endogenous natriuretic factor: LLU-alpha.
Authors: Wechter W J , Kantoci D , Murray E D , D'Amico D C , Jung M E , Wang W H
Published: July 2002

For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-alpha, which proved to be 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman, presumably a metabolite of gamma-tocopherol. Both natural LLU-alpha and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-alpha is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.
Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.
Authors: Jung K H , Spudich J L
Published: July 2002

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.
Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.
Authors: Zünd G , Nelson D P , Neufeld E J , Dzus A L , Bischoff J , Mayer J E , Colgan S P
Published: July 2002

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.
Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.
Authors: van Veen H W , Venema K , Bolhuis H , Oussenko I , Kok J , Poolman B , Driessen A J , Konings W N
Published: July 2002

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.
p300 is a component of an estrogen receptor coactivator complex.
Authors: Hanstein B , Eckner R , DiRenzo J , Halachmi S , Liu H , Searcy B , Kurokawa R , Brown M
Published: July 2002

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates expression of target genes in response to estrogen in concert with other cellular signaling pathways. This suggests that the mechanism by which ER transmits an activating signal to the general transcription machinery may include factors that integrate these diverse signals. We have previously characterized the estrogen receptor-associated protein, ERAP160, as a factor that complexes with ER in an agonist-dependent manner. We have now found that the transcriptional coactivator p300 associates with agonist bound ER and augments ligand-dependent activation by ER. Our studies show that an ER coactivator complex involves a direct hormone-dependent interaction between ER and ERAP160, resulting in the recruitment of p300. In addition, antibodies directed against the cloned steroid receptor coactivator 1 (SRC1) recognize ERAP160. The known role of p300 in multiple signal transduction pathways, including those involving the second messenger cAMP, suggests p300 functions as a point of integration between ER and these other pathways.
Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein
Authors: Kessler Paul D. , Podsakoff Gregory M. , Chen Xiaojuan , McQuiston Susan A. , Colosi Peter C. , Matelis Laura A. , Kurtzman Gary J. , Byrne Barry J.
Published: July 2002

Somatic gene therapy has been proposed as a means to achieve
systemic delivery of therapeutic proteins. However, there is limited
evidence that current methods of gene delivery can practically achieve
this goal. In this study, we demonstrate that, following a single
intramuscular administration of a recombinant adeno-associated virus
(rAAV) vector containing the β-galactosidase (AAV-lacZ) gene into
adult BALB/c mice, protein expression was detected in myofibers for
at least 32 weeks. A single intramuscular administration of an AAV
vector containing a gene for human erythropoietin (AAV-Epo) into mice
resulted in dose-dependent secretion of erythropoietin and
corresponding increases in red blood cell production that persisted for
up to 40 weeks. Primary human myotubes transduced
in
vitro
with the AAV-Epo vector also showed dose-dependent
production of Epo. These results demonstrate that rAAV vectors are able
to transduce skeletal muscle and are capable of achieving sustained
expression and systemic delivery of a therapeutic protein following a
single intramuscular administration. Gene therapy using AAV vectors may
provide a practical strategy for the treatment of inherited and
acquired protein deficiencies.
Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus
Authors: Schulte Anke M. , Lai Shoupeng , Kurtz Andreas , Czubayko Frank , Riegel Anna T. , Wellstein Anton
Published: July 2002

Retroviral elements are found in abundance throughout the human
genome but only rarely have alterations of endogenous genes
by retroviral insertions been described. Herein we report that a human
endogenous retrovirus (HERV) type C is inserted in the
human growth factor gene pleiotrophin (PTN) between the 5′ untranslated
and the coding region. This insert in the human genome expands the
region relative to the murine gene. Studies with promoter-reporter
constructs show that the HERV insert in the human PTN gene generates an
additional promoter with trophoblast-specific activity. Due to this
promoter function, fusion transcripts between HERV and the open reading
frame of PTN (HERV-PTN) were detected in all normal human trophoblast
cell cultures as early as 9 weeks after gestation
(
n
= 7) and in all term placenta tissues
(
n
= 5) but not in other normal adult tissues.
Furthermore, only trophoblast-derived choriocarcinoma cell lines
expressed HERV-PTN mRNA whereas tumor cell lines derived from the
embryoblast (teratocarcinoma) or from other lineages failed to do so.
We investigated the significance of HERV-PTN mRNA in a choriocarcinoma
model by targeting this transcript with ribozymes and found that the
depletion of HERV-PTN mRNA prevents human choriocarcinoma growth,
invasion, and angiogenesis in mice. This suggests that the
tissue-specific expression of PTN due to the HERV insertion in the
human genome supports the highly aggressive growth of human
choriocarcinoma and possibly of the human trophoblast.
Independent evolution of the prochlorophyte and green plant chlorophyll
a/b
light-harvesting proteins
Authors: Roche J. La , van der Staay G. W. M. , Partensky F. , Ducret A. , Aebersold R. , Li R. , Golden S. S. , Hiller R. G. , Wrench P. M. , Larkum A. W. D. , Green B. R.
Published: July 2002

The prochlorophytes are oxygenic prokaryotes differing from
other cyanobacteria by the presence of a light-harvesting system
containing both chlorophylls (Chls)
a
and
b
and by the absence of phycobilins. We demonstrate here
that the Chl
a/b
binding proteins from all three known
prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl
a
-binding protein induced by iron starvation, and to
CP43, a constitutively expressed Chl
a
antenna protein
of photosystem II. The prochlorophyte Chl
a/b
protein
(
pcb
) genes do not belong to the extended gene family
encoding eukaryotic Chl
a/b
and Chl
a/c
light-harvesting proteins. Although higher plants
and prochlorophytes share common pigment complements, their
light-harvesting systems have evolved independently.
Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.
Authors: Miyake S , Makimura M , Kanegae Y , Harada S , Sato Y , Takamori K , Tokuda C , Saito I
Published: July 2002

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.
In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.
Authors: Sundstedt A , Sigvardsson M , Leanderson T , Hedlund G , Kalland T , Dohlsten M
Published: July 2002

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.
Viruses, cytokines, antigens, and autoimmunity.
Authors: Gianani R , Sarvetnick N
Published: July 2002

To explain the pathogenesis of autoimmunity, we hypothesize that following an infection the immune response spreads to tissue-specific autoantigens in genetically predisposed individuals eventually determining progression to disease. Molecular mimicry between viral and self antigens could, in some instances, initiate autoimmunity. Local elicitation of inflammatory cytokines following infection probably plays a pivotal role in determining loss of functional tolerance to self autoantigens and the destructive activation of autoreactive cells. We also describe the potential role of interleukin 10, a powerful B-cell activator, in increasing the efficiency of epitope recognition, that could well be crucial to the progression toward disease.
Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping.
Authors: Wilcox P L , Amerson H V , Kuhlman E G , Liu B H , O'Malley D M , Sederoff R R
Published: July 2002

Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible.
Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage
Authors: Rieder Conly L. , Khodjakov Alexey , Paliulis Leocadia V. , Fortier Tina M. , Cole Richard W. , Sluder Greenfield
Published: July 2002

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK
1
cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK
1
cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles
x̄
= 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase
x̄
= 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.
Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity
Authors: Maurel Christophe , Tacnet Frédérique , Güclü Josette , Guern Jean , Ripoche Pierre
Published: July 2002

The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of ≥4- to 5-fold and reduced contamination by other cellular membranes. For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-flow light scattering. Osmotic water permeability coefficients (
P
f
) of 6.1 ± 0.2 and 7.6 ± 0.9 μm⋅s
−1
were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (
E
a
; 13.7 ± 1.0 and 13.4 ± 1.4 kcal⋅mol
−1
, respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix. In contrast, water transport in TP vesicles purified by FFE was characterized by (
i
) a 100-fold higher
P
f
of 690 ± 35 μm⋅s
−1
, (
ii
) a reduced
E
a
of 2.5 ± 1.3 kcal⋅mol
−1
, and (
iii
) a reversible inhibition by mercuric chloride, up to 83% at 1 mM. These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane. A high TP
P
f
suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm. Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.