bluuebunny/crossref_metadata_2025 huggingface dataset:
GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors.
Authors: Hong H , Kohli K , Trivedi A , Johnson D L , Stallcup M R
Published: July 2002

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.
Authors: Xu S , Robbins D J , Christerson L B , English J M , Vanderbilt C A , Cobb M H
Published: July 2002

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.
Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.
Authors: Takayama H , La Rochelle W J , Anver M , Bockman D E , Merlino G
Published: July 2002

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.
Structural analysis of p185c-neu and epidermal growth factor receptor tyrosine kinases: oligomerization of kinase domains.
Authors: Murali R , Brennan P J , Kieber-Emmons T , Greene M I
Published: July 2002

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Here we analyze potential interactions of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization.
Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors.
Authors: Silber J R , Blank A , Bobola M S , Mueller B A , Kolstoe D D , Ojemann G A , Berger M S
Published: July 2002

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.
Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.
Authors: Bories J C , Demengeot J , Davidson L , Alt F W
Published: July 2002

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.
Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport.
Authors: Krysan P J , Young J C , Tax F , Sussman M R
Published: July 2002

The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.
Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.
Authors: Wang R , Kobayashi R , Bishop J M
Published: July 2002

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.
Role for cells in the presupplementary motor area in updating motor plans.
Authors: Shima K , Mushiake H , Saito N , Tanji J
Published: July 2002

Two motor areas are known to exist in the medial frontal lobe of the cerebral cortex of primates, the supplementary motor area (SMA) and the presupplementary motor area (pre-SMA). We report here on an aspect of cellular activity that characterizes the pre-SMA. Monkeys were trained to perform three different movements sequentially in a temporal order. The correct order was planned on the basis of visual information before its execution. A group of pre-SMA cells (n = 64, 25%) were active during a process when monkeys were required to discard a current motor plan and develop a plan appropriate for the next orderly movements. Such activity was not common in the SMA and not found in the primary motor cortex. Our data suggest a role of pre-SMA cells in updating motor plans for subsequent temporally ordered movements.
Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.
Authors: Jardetzky T S , Brown J H , Gorga J C , Stern L J , Urban R G , Strominger J L , Wiley D C
Published: July 2002

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.
A combined kinetic and modeling study of the catalytic center subsites of human angiogenin.
Authors: Russo N , Acharya K R , Vallee B L , Shapiro R
Published: July 2002

Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B1 pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B1. Although the B2 site that preferentially binds purines on the 3' side of B1 is also weak, its associated phosphate subsites make substantial contributions: both 3',5'-ADP and 5'-ADP have Ki values 6-fold lower than for 5'-AMP, and adding a 3'-phosphate to the substrate CpA increases Kcat/Km by 9-fold. Thus Ang has a functional P2 site on the 3' side of B2 and a site for a second phosphate on the 5' side of B2. Modeling of an Ang-d(ApTpApA) complex suggested that Arg-5 forms part of the P2 site and that a 2'-phosphate might bind more tightly than a 3'-phosphate. Both predictions were confirmed kinetically. The subsite map obtained by this combined approach indicated that 5'-diphosphoadenosine 2'-phosphate might be a more potent inhibitor than any of the nucleotides tested thus far. Indeed, its Ki value of 150 microM is 50-fold lower than that for the best nucleotide previously reported and 400-fold lower than the Km for the best dinucleotide substrate. This compound may serve as a suitable starting point for the eventual design of tight-binding inhibitors of Ang as antiangiogenic agents for human therapy.
Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.
Authors: Blyn L B , Swiderek K M , Richards O , Stahl D C , Semler B L , Ehrenfeld E
Published: July 2002

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.
Resistance gene analogs are conserved and clustered in soybean.
Authors: Kanazin V , Marek L F , Shoemaker R C
Published: July 2002

Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.
Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation.
Authors: Popov S , Dubrovsky L , Lee M A , Pennathur S , Haffar O , aL-Abed Y , Tonge P , Ulrich P , Rexach M , Blobel G , Cerami A , Bukrinsky M
Published: July 2002

HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to karyopherin alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.
Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides
Authors: Sierakowska Halina , Sambade Maria J. , Agrawal Sudhir , Kole Ryszard
Published: July 2002

In one form of β-thalassemia, a genetic blood disorder, a
mutation in intron 2 of the β-globin gene (IVS2-654) causes aberrant
splicing of β-globin pre-mRNA and, consequently, β-globin
deficiency. Treatment of mammalian cells stably expressing the IVS2-654
human β-globin gene with antisense oligonucleotides targeted at the
aberrant splice sites restored correct splicing in a dose-dependent
fashion, generating correct human β-globin mRNA and polypeptide. Both
products persisted for up to 72 hr posttreatment. The oligonucleotides
modified splicing by a true antisense mechanism without overt
unspecific effects on cell growth and splicing of other pre-mRNAs. This
novel approach in which antisense oligonucleotides are used to restore
rather than to down-regulate the activity of the target gene is
applicable to other splicing mutants and is of potential clinical
interest.
γ-Aminobutyric acid type B receptor-dependent burst-firing in thalamic neurons: A dynamic clamp study
Authors: Ulrich Daniel , Huguenard John R.
Published: July 2002

Synchronized network responses in thalamus depend on phasic
inhibition originating in the thalamic reticular nucleus (nRt) and are
mediated by the neurotransmitter γ-aminobutyric acid (GABA). A
suggested role for intra-nRt connectivity in inhibitory phasing remains
controversial. Recently, functional GABA type B (GABA
B
)
receptors were demonstrated on nRt cells, and the slow time course of
the GABA
B
synaptic response seems ideally suited to
deinactivate low-threshold calcium channels. This promotes burst
firing, a characteristic feature of synchronized responses. Here we
investigate GABA
B
-mediated rebound burst firing in thalamic
cells. Whole-cell current-clamp recordings were obtained from nRt cells
and somatosensory thalamocortical relay cells in rat brain slices.
Synthetic GABA
B
inhibitory postsynaptic potentials,
generated by a hybrid computer–neuron synapse (dynamic clamp),
triggered rebound low-threshold calcium spikes in both cell types when
peak inhibitory postsynaptic potential hyperpolarization was greater
than −92 mV. The threshold inhibitory postsynaptic potential
conductance for rebound burst generation was comparable in nRt (7 nS)
and thalamocortical (5 nS) cells. However, burst onset in nRt (1 s) was
considerably delayed compared with thalamocortical (0.6 s) cells. Thus,
GABA
B
inhibitory postsynaptic potentials can elicit
low-threshold calcium spikes in both relay and nRt neurons, but the
resultant oscillation frequency would be faster for
thalamocortical–nRt networks (3 Hz) than for nRt–nRt networks (1–2
Hz). We conclude, therefore, that fast (>2 Hz)
GABA
B
-dependent thalamic oscillations are maintained
primarily by reciprocal connections between excitatory and inhibitory
cells. These findings further indicate that when oscillatory neural
networks contain both recurrent and reciprocal inhibition, then
distinct population frequencies may result when one or the other type
of inhibition is favored.
Time-resolved optical diffusion tomographic image reconstruction in highly scattering turbid media
Authors: Cai W. , Das B. B. , Liu Feng , Zevallos M. , Lax M. , Alfano R. R.
Published: July 2002

The image of an object hidden in highly scattering media was reconstructed using a fast, noise-resistant algorithm newly applied to diffusion tomography. A pulsed light source producing scattered and transmitted light is examined at multiple times. Multiple source detector pairs around the medium are used to obtain data in many different directions. An inverse scattering algorithm with nonuniform regularization achieves rapid inversion convergence.
Misconceptions about mitochondria and mammalian fertilization: Implications for theories on human evolution
Authors: Ankel-Simons Friderun , Cummins Jim M.
Published: July 2002

In vertebrates, inheritance of mitochondria is thought to be
predominantly maternal, and mitochondrial DNA analysis has become a
standard taxonomic tool. In accordance with the prevailing view of
strict maternal inheritance, many sources assert that during
fertilization, the sperm tail, with its mitochondria, gets excluded
from the embryo. This is incorrect. In the majority of
mammals—including humans—the midpiece mitochondria can be identified
in the embryo even though their ultimate fate is unknown. The
“missing mitochondria” story seems to have survived—and
proliferated—unchallenged in a time of contention between hypotheses
of human origins, because it supports the “African Eve” model of
recent radiation of
Homo sapiens
out of Africa. We will
discuss the infiltration of this mistake into concepts of mitochondrial
inheritance and human evolution.
Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)
Authors: Russo James J. , Bohenzky Roy A. , Chien Ming-Cheng , Chen Jing , Yan Ming , Maddalena Dawn , Parry J. Preston , Peruzzi Daniela , Edelman Isidore S. , Chang Yuan , Moore Patrick S.
Published: July 2002

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase,bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.
Regulation of product chain length by isoprenyl diphosphate synthases
Authors: Tarshis L. C. , Proteau Philip J. , Kellogg Brenda A. , Sacchettini James C. , Poulter C. Dale
Published: July 2002

An analysis of the x-ray structure of homodimeric avian
farnesyl diphosphate synthase (geranyltransferase, EC
2.5.1.10
) coupled
with information about conserved amino acids obtained from a sequence
alignment of 35 isoprenyl diphosphate synthases that synthesize
farnesyl (C
15
), geranylgeranyl (C
20
), and
higher chain length isoprenoid diphosphates suggested that the side
chains of residues corresponding to F112 and F113 in the avian enzyme
were important for determining the ultimate length of the hydrocarbon
chains. This hypothesis was supported by site-directed mutagenesis to
transform wild-type avian farnesyl diphosphate synthase (FPS) into
synthases capable of producing geranylgeranyl diphosphate (F112A),
geranylfarnesyl (C
25
) diphosphate (F113S), and longer chain
prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure
of the F112A/F113S mutant in the apo state and with allylic
substrates bound produced the strongest evidence that these mutations
caused the observed change in product specificity by directly altering
the size of the binding pocket for the growing isoprenoid chain in the
active site of the enzyme. The proposed binding pocket in the apo
mutant structure was increased in depth by 5.8 Å as compared with that
for the wild-type enzyme. Allylic diphosphates were observed in the
holo structures, bound through magnesium ions to the aspartates of the
first of two conserved aspartate-rich sequences (D117–D121), with the
hydrocarbon tails of all the ligands growing down the hydrophobic
pocket toward the mutation site. A model was constructed to show how
the growth of a long chain prenyl product may proceed by creation of a
hydrophobic passageway from the FPS active site to the outside surface
of the enzyme.
Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy
Authors: Lee YoungJoo , Gorski Jack
Published: July 2002

The activation of the silent endogenous progesterone
receptor (PR) gene by 17-β-estradiol (E
2
) in cells stably
transfected with estrogen receptor (ER) was used as a model system to
study the mechanism of E
2
-induced transcription. The time
course of E
2
-induced PR transcription rate was determined
by nuclear run-on assays. No marked effect on specific PR gene
transcription rates was detected at 0 and 1 h of E
2
treatment. After 3 h of E
2
treatment, the PR mRNA
synthesis rate increased 2.0- ± 0.2-fold and continued to increase to
3.5- ± 0.4-fold by 24 h as compared with 0 h. The
transcription rate increase was followed by PR mRNA accumulation. No PR
mRNA was detectable at 0, 1, and 3 h of E
2
treatment.
PR mRNA accumulation was detected at 6 h of E
2
treatment and continued to accumulate until 18 h, the longest time
point examined. Interestingly, this slow and gradual transcription rate
increase of the endogenous PR gene did not parallel binding
of E
2
to ER, which was maximized within 30 min.
Furthermore, the E
2
–ER level was down-regulated to 15% at
3 h as compared with 30 min of E
2
treatment and
remained low at 24 h of E
2
exposure. These paradoxical
observations indicate that E
2
-induced transcription
activation is more complicated than just an association of the occupied
ER with the transcription machinery.
Fusion of the transcription factor
TFE3
gene to a novel gene,
PRCC
, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas
Authors: Weterman Marian A. J. , Wilbrink Monique , van Kessel Ad Geurts
Published: July 2002

The (X;1)(p11;q21) translocation is a recurrent chromosomal
abnormality in a subset of human papillary renal cell carcinomas, and
is sometimes the sole cytogenetic abnormality present. Via positional
cloning, we were able to identify the genes involved. The translocation
results in a fusion of the transcription factor
TFE3
gene on the X chromosome to a novel gene, designated
PRCC
, on chromosome 1. Through this fusion, reciprocal
translocation products are formed, which are both expressed in
papillary renal cell carcinomas.
PRCC
is ubiquitously
expressed in normal adult and fetal tissues and encodes a putative
protein of 491 aa with a relatively high content of prolines. No
relevant homologies with known sequences at either the DNA or the
protein level were found.
The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.
Authors: Ménard R , Prévost M C , Gounon P , Sansonetti P , Dehio C
Published: July 2002

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.
Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.
Authors: Schröder S , Lohse M J
Published: July 2002

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.
High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.
Authors: Singer S M , Umetsu D T , McDevitt H O
Published: July 2002

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.
Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis.
Authors: Liblau R S , Tisch R , Shokat K , Yang X , Dumont N , Goodnow C C , McDevitt H O
Published: July 2002

The mechanism by which tolerance is induced via systemic administration of high doses of aqueous antigen has been analyzed by using mice transgenic for a T-cell receptor specific for the influenza virus hemagglutinin (HA) peptide comprising amino acids 126-138. After intravenous injection of 750 (but not 75) micrograms of HA peptide, a state of hyporesponsiveness was rapidly induced. In the thymus, in situ apoptosis in the cortex and at the corticomedullary junction was responsible for a synchronous and massive deletion of CD4+ CD8+ thymocytes. In secondary lymphoid organs, HA-reactive T cells were initially activated but were hyporesponsive at the single cell level. After 3 days, however, those cells were rapidly deleted, at least partially, through an apoptotic process. Therefore, both thymic and peripheral apoptosis, in addition to T-cell receptor desensitization, contribute to high-dose tolerance.
Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.
Authors: Mindell D P
Published: July 2002

Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host-species shifts for PIV1s.
Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.
Authors: Hecht S J , Stedman K E , Carlson J O , DeMartini J C
Published: July 2002

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.
Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.
Authors: Luebke A E , Dahl G P , Roos B A , Dickerson I M
Published: July 2002

An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.
Differences in the RNA binding sites of iron regulatory proteins and potential target diversity.
Authors: Butt J , Kim H Y , Basilion J P , Cohen S , Iwai K , Philpott C C , Altschul S , Klausner R D , Rouault T A
Published: July 2002

Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system.
Functional antioxidant responsive elements
Authors: Wasserman Wyeth W. , Fahl William E.
Published: July 2002

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione
S
-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE “core” sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE–binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.
Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract
Authors: Goodman F. R. , Mundlos S. , Muragaki Y. , Donnai D. , Giovannucci-Uzielli M. L. , Lapi E. , Majewski F. , McGaughran J. , McKeown C. , Reardon W. , Upton J. , Winter R. M. , Olsen B. R. , Scambler P. J.
Published: July 2002

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation. Typical cases have 3/4 finger and 4/5 toe syndactyly, with a duplicated digit in the syndactylous web, but incomplete penetrance and variable expressivity are common. The condition has recently been shown to be caused by expansions of an imperfect trinucleotide repeat sequence encoding a 15-residue polyalanine tract in HOXD13. We have studied 16 new and 4 previously published SPD families, with between 7 and 14 extra residues in the tract, to analyze the molecular basis for the observed variation in phenotype. Although there is no evidence of change in expansion size within families, even over six generations, there is a highly significant increase in the penetrance and severity of phenotype with increasing expansion size, affecting both hands (
P
= 0.012) and feet (
P
< 0.00005). Affected individuals from a family with a 14-alanine expansion, the largest so far reported, all have a strikingly similar and unusually severe limb phenotype, involving the first digits and distal carpals. Affected males from this family also have hypospadias, not previously described in SPD, but consistent with HOXD13 expression in the developing genital tubercle. The remarkable correlation between phenotype and expansion size suggests that expansion of the tract leads to a specific gain of function in the mutant HOXD13 protein, and has interesting implications for the role of polyalanine tracts in the control of transcription.
Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone
Authors: Morley John E. , Kaiser Fran , Raum William J. , Perry H. Mitchell , Flood James F. , Jensen Judy , Silver Andrew J. , Roberts Eugene
Published: July 2002

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (
r
> −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive
and
physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.