bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors
Authors: Yeh Brian K. , Igarashi Makoto , Eliseenkova Anna V. , Plotnikov Alexander N. , Sher Ifat , Ron Dina , Aaronson Stuart A. , Mohammadi Moosa
Published: March 2003

Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb (“b”) and IIIc (“c”), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.
Sex- and age-dependent human transcriptome variability: Implications for chronic heart failure
Authors: Boheler Kenneth R. , Volkova Maria , Morrell Christopher , Garg Rahul , Zhu Yi , Margulies Kenneth , Seymour Anne-Marie , Lakatta Edward G.
Published: March 2003

Heart failure (HF) is the end result of progressive and diverse biological adaptations within the diseased myocardium. We used cDNA microarrays and quantitative PCR to examine the transcriptomes of 38 left ventricles from failing and nonfailing human myocardium. After identification of a pool of putative HF-responsive candidate genes by microarrays on seven nonfailing and eight failing hearts, we used quantitative PCR and a general linear statistical model in a larger sample set (
n
= 34) to validate and examine the role of contributing biological variables (age and sex). We find that most HF-candidate genes (transcription factors, Cebpb, Npat; signaling molecules, Map2k3, Map4k5; extracellular matrix proteins, Lum, Cola1; and metabolic enzymes, Mars) demonstrated significant changes in gene expression; however, the majority of differences among samples depended on variables such as sex and age, and not on HF alone. Some HF-responsive gene products also demonstrated highly significant changes in expression as a function of age and/or sex, but independent of HF (Ngp1, Cd163, and Npat). These results emphasize the need to account for biological variables (HF, sex and age interactions) to elucidate genomic correlates that trigger molecular pathways responsible for the progression of HF syndromes.
Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry
Authors: Kalkum Markus , Lyon Gholson J. , Chait Brian T.
Published: March 2003

A method is presented for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum-sensing peptides. The procedure, based on targeted multistage MS, uses a novel matrix-assisted laser desorption/ionization-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, and chemical noise) that impair observation of low abundance peptides from complex biological matrixes. Here, secreted peptides that are hypothesized to be present in the supernatant, but that may not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multistage MS. Highly specific fragmentation signatures enable unambiguous identification of the peptides of interest and differentiation of the signals from the background. As examples, we demonstrate the rapid (<1 min) determination of the mating type of cells in colonies of
Saccharomyces cerevisiae
and the elucidation of autoinducing peptides (AIPs) from supernatants of pathogenic Staphylococci. We confirm the primary structures of the
agrD
encoded cyclic AIPs of
Staphylococcus aureus
for groups I, II, and IV and provide direct evidence that the native group-III AIP is a heptapeptide (INCDFLL). We also show that the homologous peptide from
Staphylococcus intermedius
is a nonapeptide (RIPTSTGFF) with a lactone ring formed through condensation of the serine side chain with the C terminus of the peptide. This is the first demonstration of cyclization in a staphylococcal AIP that occurs via lactone formation. These examples demonstrate the analytical power of the present procedure for characterizing secreted peptides and its potential utility for identifying microorganisms.
Probing the kinesin reaction cycle with a 2D optical force clamp
Authors: Block Steven M. , Asbury Charles L. , Shaevitz Joshua W. , Lang Matthew J.
Published: March 2003

With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADP/P
i
release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.
Transgenerational inheritance of epigenetic states at the murine
Axin

Fu

allele occurs after maternal and paternal transmission
Authors: Rakyan Vardhman K. , Chong Suyinn , Champ Marnie E. , Cuthbert Peter C. , Morgan Hugh D. , Luu Keith V. K. , Whitelaw Emma
Published: March 2003

Phenotypic variation that cannot be explained by genetic or environmental heterogeneity has intrigued geneticists for decades. The molecular basis of this phenomenon, however, is largely a mystery.
Axin-fused
(

Axin
Fu

), first identified in 1937, is a classic example of a mammalian allele displaying extremely variable expression states. Here we demonstrate that the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotransposon within

Axin
Fu

and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype. Furthermore, the epigenetic state at

Axin
Fu

can be inherited transgenerationally after both maternal and paternal transmission. This is in contrast to epigenetic inheritance at the murine agouti-viable yellow (

A
vy

) allele, which occurs through the female only. Unlike the egg, the sperm contributes very little (if any) cytoplasm to the zygote, and therefore paternal inheritance at

Axin
Fu

argues against the possibility that the effects are due to cytoplasmic or metabolic influences. Consistent with the idea of transgenerational inheritance of epigenetic marks, we find that the methylation state of

Axin
Fu

in mature sperm reflects the methylation state of the allele in the somatic tissue of the animal, suggesting that it does not undergo epigenetic reprogramming during gametogenesis. Finally, we show that epigenetic inheritance is influenced by strain background. These findings enable us to propose a model for transgenerational epigenetic inheritance in mammals.
Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation
Authors: Xiao Bing , Spencer James , Clements Adrienne , Ali-Khan Nadeem , Mittnacht Sibylle , Broceño Cristina , Burghammer Manfred , Perrakis Anastassis , Marmorstein Ronen , Gamblin Steven J.
Published: March 2003

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409–426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the “marked box” region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2/cyclin D/E. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.
Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint
Authors: Majka Jerzy , Burgers Peter M. J.
Published: March 2003

The
Saccharomyces cerevisiae
Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17/3/1 showed no detectable exonuclease activity.
De novoprotein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination
Authors: Doi Tomomitsu , Kinoshita Kazuo , Ikegawa Masaya , Muramatsu Masamichi , Honjo Tasuku
Published: March 2003

Activation-induced cytidine deaminase (AID) is required for class-switch recombination (CSR), somatic hypermutation, and gene conversion of Ig genes. Although AID has sequence similarity to an RNA-editing enzyme Apobec-1, how AID functions in CSR and somatic hypermutation is unknown. Because involvement of RNA-editing but not DNA-editing in CSR requiresde novoprotein synthesis after AID expression, we examined whether protein synthesis inhibitors could block CSR in the presence of the AID activity. For this purpose we constructed AID fused with the hormone-binding domain of the estrogen receptor (AID-ER), which was introduced into AID-deficient spleen B cells. When such transfectants were treated with an estrogen analogue, 4-hydroxytamoxifen (OHT), CSR was induced within 1 h. Cycloheximide or puromycin drastically suppressed OHT-induced CSR in AID-ER expressing AID−/− B cells when added 1 h before OHT but not after OHT, suggesting thatde novoprotein synthesis is required for an event downstream to AID expression in CSR. The results lend the weight to RNA-editing hypothesis for the function of AID.
Androgen-induced recruitment of RNA polymerase II to a nuclear receptor–p160 coactivator complex
Authors: Louie Maggie C. , Yang Hong Qiong , Ma Ai-Hong , Xu Wei , Zou June X. , Kung Hsing-Jien , Chen Hong-Wu
Published: March 2003

The androgen receptor, like other nuclear receptors, activates target genes by binding to hormone-responsive enhancers. Here we demonstrate that androgen induces robust recruitment of androgen receptor, members of the p160 coactivator family, and CREB-binding protein/p300 specifically at the distant enhancer of prostate-specific antigen (PSA) gene. Unexpectedly, we found that RNA polymerase II (Pol II) is directly recruited to the enhancer in a hormone-dependent manner, independent of the proximal promoter, and that the isolated PSA enhancer can mediate efficient androgen induction of transcription. Inhibition of the Pol II carboxyl-terminal domain kinase activity with low concentrations of flavopiridol blocks Pol II transfer from the enhancer to the promoter and selectively abolishes PSA induction by androgen. Moreover, elevated levels of the p160 coactivator ACTR/AIB1 increase both androgen-dependent and -independent PSA expression, by facilitating Pol II recruitment to the enhancer. These results support a model in which nuclear receptors and their coactivators mediate hormone induction by serving as a staging platform for Pol II recruitment.
Structure of a cholesterol-binding protein deficient in Niemann–Pick type C2 disease
Authors: Friedland Natalia , Liou Heng-Ling , Lobel Peter , Stock Ann M.
Published: March 2003

Niemann–Pick disease type C2 (NP-C2) is a fatal hereditary disease characterized by accumulation of low-density lipoprotein-derived cholesterol in lysosomes. Here we report the 1.7-Å resolution crystal structure of the cholesterol-binding protein deficient in this disease, NPC2, and the characterization of its ligand binding properties. Human NPC2 binds the cholesterol analog dehydroergosterol with submicromolar affinity at both acidic and neutral pH. NPC2 has an Ig-like fold stabilized by three disulfide bonds. The structure of the bovine protein reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of ≈160 Å
3
. We propose that this region represents the incipient cholesterol-binding site that dilates to accommodate an ≈740-Å
3
cholesterol molecule.
Optimally sparse representation in general (nonorthogonal) dictionaries via ℓ
1
minimization
Authors: Donoho David L. , Elad Michael
Published: March 2003

Given a dictionary D = {

d

k

} of vectors

d

k

, we seek to represent a signal

S

as a linear combination

S

= ∑

k

γ(
k
)

d

k

, with scalar coefficients
γ
(
k
). In particular, we aim for the sparsest representation possible. In general, this requires a combinatorial optimization process. Previous work considered the special case where D is an overcomplete system consisting of exactly two orthobases and has shown that, under a condition of mutual incoherence of the two bases, and assuming that

S

has a sufficiently sparse representation, this representation is unique and can be found by solving a convex optimization problem: specifically, minimizing the ℓ
1
norm of the coefficients γ̱. In this article, we obtain parallel results in a more general setting, where the dictionary D can arise from two or several bases, frames, or even less structured systems. We sketch three applications: separating linear features from planar ones in 3D data, noncooperative multiuser encoding, and identification of over-complete independent component models.
In vivo
identification of human cortical areas using high-resolution MRI: An approach to cerebral structure–function correlation
Authors: Walters Nathan B. , Egan Gary F. , Kril Jillian J. , Kean Michael , Waley Patricia , Jenkinson Mark , Watson John D. G.
Published: March 2003

Understanding the relationship between the structural and functional organization of the human brain is one of the most important goals of neuroscience. Individual variability in brain structure means that it is essential to obtain this information from the same subject. To date, this has been almost impossible. Even though noninvasive functional imaging techniques such as functional MRI (fMRI) are now commonplace, there is no complementary noninvasive structural technique. We present an
in vivo
method of examining the detailed neuroanatomy of any individual, which can then be correlated with that individual's own functional results. This method utilizes high-resolution structural MRI to identify distinct cortical regions based on cortical lamination structure. We demonstrate that the observed MR lamination patterns relate to myeloarchitecture through a correlation of histology with MRI.
In vivo
high-resolution MRI studies identify striate cortex, as well as visual area V5, in four individuals, as defined by using fMRI. The anatomical identification of a cortical area (V5/MT) outside of striate cortex is a significant advance, proving it possible to identify extra-striate cortical areas and demonstrating that
in vivo
structural mapping of the human cerebral cortex is possible.
Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded
Authors: Denning Daniel P. , Patel Samir S. , Uversky Vladimir , Fink Anthony L. , Rexach Michael
Published: March 2003

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The
Saccharomyces cerevisiae
NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of
S. cerevisiae
FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of “natively unfolded” proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered
in situ
within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.
Single-locus heterotic effects and dominance by dominance interactions can adequately explain the genetic basis of heterosis in an elite rice hybrid
Authors: Hua Jinping , Xing Yongzhong , Wu Weiren , Xu Caiguo , Sun Xinli , Yu Sibin , Zhang Qifa
Published: March 2003

The genetic basis of heterosis of an elite rice hybrid was investigated by using an “immortalized F2” population produced by randomly permutated intermating of 240 recombinant inbred lines from a cross between the parents of Shanyou 63, the most widely cultivated hybrid in China. Measurements of heterosis for crosses in the immortalized F2population were obtained from replicated field trials over 2 years by inter-planting the hybrids with the parental recombinant inbred lines. The analyses were conducted making use of a linkage map comprising 231 segregating molecular marker loci covering the entire rice genome. Heterotic effects were detected at 33 loci for the four traits with modified composite interval mapping. The heterotic loci showed little overlap with quantitative trait loci for trait performance, suggesting that heterosis and trait performance may be conditioned by different sets of loci. Large numbers of digenic interactions were resolved by using two-way ANOVA and confirmed by randomization tests. All kinds of genetic effects, including partial-, full-, and overdominance at single-locus level and all three forms of digenic interactions (additive by additive, additive by dominance, and dominance by dominance), contributed to heterosis in the immortalized F2population, indicating that these genetic components were not mutually exclusive in the genetic basis of heterosis. Heterotic effects at the single-locus level, in combination with the marginal advantages of double heterozygotes caused by dominance by dominance interaction at the two-locus level could adequately explain the genetic basis of heterosis in Shanyou 63. These results may help reconcile the century-long debate concerning the genetic basis of heterosis.
Transgene produces massive overexpression of human β-glucuronidase in mice, lysosomal storage of enzyme, and strain-dependent tumors
Authors: Vogler Carole , Galvin Nancy , Levy Beth , Grubb Jeffery , Jiang Jinxing , Zhou Xiao Yan , Sly William S.
Published: March 2003

β-Glucuronidase (GUSB) is a lysosomal enzyme important in the normal step-wise degradation of glycosaminoglycans. Deficiency of GUSB causes the lysosomal storage disease mucopolysaccharidosis VII (MPS VII, Sly disease). Affected patients have widespread progressive accumulation of β-glucuronide-containing glycosaminoglycans in lysosomes. Enzyme replacement, bone marrow transplantation, and gene therapy can correct lysosomal storage in the MPS VII mouse model. Gene therapy in MPS VII patients and animals may result in massive overexpression of GUSB in individual tissues, and the toxicity of such overexpression is incompletely investigated. To gain insight into the effect of massive overexpression of GUSB, we established 19 transgenic mouse lines, two of which expressed very high levels of human GUSB in many tissues. The founder overexpressing mice had from >100- to several thousand-fold increases in tissue and serum GUSB. The enzyme expression in most tissues decreased in subsequent generations in one line, and expression in liver and marrow fell in subsequent generations of the other. Both lines had morphologically similar widespread lysosomal storage of GUSB and secondary elevations of other lysosomal enzymes, a finding characteristic of lysosomal storage disease. One line developed tumors, and one did not. These transgenic models show that massive overexpression of a lysosomal enzyme can be associated with dramatic morphological alterations, which, at least in one of the two lines, had little clinical consequence. For the other transgenic line, the high frequency of tumor development in F
2
FVB progeny suggests that the vector used to generate the transgenic lines has an integration site-dependent potential to be oncogenic, at least in this strain background.
T cell receptor (TCR) α/δ locus enhancer identity and position are critical for the assembly of TCR δ and α variable region genes
Authors: Bassing Craig H. , Tillman Robert E. , Woodman Barbara B. , Canty David , Monroe Robert J. , Sleckman Barry P. , Alt Frederick W.
Published: March 2003

T cell receptor (TCR) δ and α variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCRδ segments are situated between TCRα segments. The TCRα enhancer (Eα) located at the 3′ end of the TCRα/δ locus functions over a long chromosomal distance to promote TCRα rearrangement and maximal TCRδ expression; whereas the TCRδ enhancer (Eδ) is located among the TCRδ segments and functions with additional element(s) to mediate TCRδ rearrangement. We used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ are critical for the developmental stage-specific assembly of TCR δ and α variable region genes. Specific replacement of Eα with Eδ, the core Eα element (EαC), or the Ig heavy chain intronic enhancer (iEμ), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCRα rearrangement beyond that observed in the absence of Eα. Therefore, the identity and full complement of Eα-binding sites are critical for promoting accessibility within the TCRα locus. In the absence of the endogenous Eδ element, specific replacement of Eα with Eδ also did not promote TCRδ rearrangement. However, deletion of intervening TCRα/δ locus sequences to restore the inserted Eδ to its normal chromosomal position relative to 5′ sequences rescued TCRδ rearrangement. Therefore, unlike Eα, Eδ lacks ability to function over the large intervening TCRα locus and/or Eδ function requires proximity to additional upstream element(s) to promote TCRδ accessibility.
Src promotes destruction of c-Cbl: Implications for oncogenic synergy between Src and growth factor receptors
Authors: Bao Jing , Gur Gal , Yarden Yosef
Published: March 2003

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.
Nonhomologous end joining and V(D)J recombination require an additional factor
Authors: Dai Y. , Kysela B. , Hanakahi L. A. , Manolis K. , Riballo E. , Stumm M. , Harville T. O. , West S. C. , Oettinger M. A. , Jeggo P. A.
Published: March 2003

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T−B−severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue inSaccharomyces cerevisiaefunctions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.
Mitotic spindle rotation and mode of cell division in the developing telencephalon
Authors: Haydar Tarik F. , Ang Eugenius , Rakic Pasko
Published: March 2003

The mode of neural stem cell division in the forebrain proliferative zones profoundly influences neocortical growth by regulating the number and diversity of neurons and glia. Long-term time-lapse multiphoton microscopy of embryonic mouse cortex reveals new details of the complex three-dimensional rotation and oscillation of the mitotic spindle before stem cell division. Importantly, the duration and amplitude of spindle movement predicts and specifies the eventual mode of mitotic division. These technological advances have provided dramatic data and insights into the kinetics of neural stem cell division by elucidating the involvement of spindle rotation in selection of the cleavage plane and the mode of neural stem cell division that together determine the size of the mammalian neocortex.
A
Saccharomyces cerevisiae
mutant with increased virulence
Authors: Wheeler Robert T. , Kupiec Martin , Magnelli Paula , Abeijon Claudia , Fink Gerald R.
Published: March 2003

Saccharomyces cerevisiae
, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of
S. cerevisiae
clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the
SSD1
gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent
ssd1
Δ/
ssd1
Δ yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages
in vitro
. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.
Differentiation, cell fusion, and nuclear fusion during
ex vivo
repair of epithelium by human adult stem cells from bone marrow stroma
Authors: Spees Jeffrey L. , Olson Scott D. , Ylostalo Joni , Lynch Patrick J. , Smith Jason , Perry Anthony , Peister Alexandra , Wang Meng Yu , Prockop Darwin J.
Published: March 2003

To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heat-shocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.
Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase IIIβ
Authors: Kwan Kelvin Y. , Moens Peter B. , Wang James C.
Published: March 2003

We report that disruption of the mouse
TOP3
β gene encoding DNA topoisomerase IIIβ, one of the two mammalian type IA DNA topoisomerases, leads to a progressive reduction in fecundity. The litter size in crosses of
top3
β
−/−
mice decreases over time and through successive generations, and this decrease seems to reflect embryonic death rather than impaired fertilization. These observations are suggestive of a gradual accumulation of chromosomal defects in germ cells lacking DNA topoisomerase IIIβ, and this interpretation is supported by the observation of a high incidence of aneuploidy in the spermatocytes of infertile
top3
β
−/−
males. Cytogenetic examination of spermatocytes of wild-type mice also indicates that DNA topoisomerase IIIβ becomes prominently associated with the asynaptic regions of the XY bivalents during pachytene, and that there is a time lag between the appearance of chromosome-bound DNA topoisomerase IIIβ and Rad51, a protein known to be involved in an early step of homologous recombination. We interpret these findings, together with the known mechanistic characteristics of different subfamilies of DNA topoisomerases, in terms of a specific role of a type IA DNA topoisomerase in the resolution of meiotic double-Holliday junctions without crossing over. This interpretation is most likely applicable to mitotic cells as well and can explain the universal presence of at least one type IA DNA topoisomerase in all organisms.
Genomewide analysis of
Drosophila
GAGA factor target genes reveals context-dependent DNA binding
Authors: van Steensel Bas , Delrow Jeffrey , Bussemaker Harmen J.
Published: March 2003

The association of sequence-specific DNA-binding factors with their cognate target sequences
in vivo
depends on the local molecular context, yet this context is poorly understood. To address this issue, we have performed genomewide mapping of
in vivo
target genes of
Drosophila
GAGA factor (GAF). The resulting list of ≈250 target genes indicates that GAF regulates many cellular pathways. We applied unbiased motif-based regression analysis to identify the sequence context that determines GAF binding. Our results confirm that GAF selectively associates with (GA)
n
repeat elements
in vivo
. GAF binding occurs in upstream regulatory regions, but less in downstream regions. Surprisingly, GAF binds abundantly to introns but is virtually absent from exons, even though the density of (GA)
n
is roughly the same. Intron binding occurs equally frequently in last introns compared with first introns, suggesting that GAF may not only regulate transcription initiation, but possibly also elongation. We provide evidence for cooperative binding of GAF to closely spaced (GA)
n
elements and explain the lack of GAF binding to exons by the absence of such closely spaced GA repeats. Our approach for revealing determinants of context-dependent DNA binding will be applicable to many other transcription factors.
A fluorescence resonance energy transfer sensor for the β-domain of metallothionein
Authors: Hong Sung-Hye , Maret Wolfgang
Published: March 2003

We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purification system, the other on the observation that one human MT isoform (1b) contains an additional cysteine at position 32. It is located in the linker region of the molecule, allowing the introduction of a probe between the two domains. An S32C mutation was introduced into hMT-2. Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the additional cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metal/thiolate clusters. MT containing only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT containing only zinc. Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal β-domain of the protein.
Microtransplantation of membranes from cultured cells to
Xenopus
oocytes: A method to study neurotransmitter receptors embedded in native lipids
Authors: Palma Eleonora , Trettel Flavia , Fucile Sergio , Renzi Massimiliano , Miledi Ricardo , Eusebi Fabrizio
Published: March 2003

The
Xenopus
oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of
Xenopus
oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing
homomeric
human neuronal α7 nicotinic acetylcholine receptors (α7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing
heteromeric
human neuronal α4β2-AcChoRs. All this makes the
Xenopus
oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells.
JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis
Authors: Lei Kui , Davis Roger J.
Published: March 2003

The c-Jun NH
2
-terminal kinase (JNK) is activated when cells are exposed to environmental stress, including UV radiation. Gene disruption studies demonstrate that JNK is essential for UV-stimulated apoptosis mediated by the mitochondrial pathway by a Bax/Bak-dependent mechanism. Here, we demonstrate that JNK phosphorylates two members of the BH3-only subgroup of Bcl2-related proteins (Bim and Bmf) that are normally sequestered by binding to dynein and myosin V motor complexes. Phosphorylation by JNK causes release from the motor complexes. These proapoptotic BH3-only proteins therefore provide a molecular link between the JNK signal transduction pathway and the Bax/Bak-dependent mitochondrial apoptotic machinery.
Immunotherapy for choroidal neovascularization in a laser-induced mouse model simulating exudative (wet) macular degeneration
Authors: Bora Puran S. , Hu Zhiwei , Tezel Tongalp H. , Sohn Jeong-Hyeon , Kang Shin Goo , Cruz Jose M. C. , Bora Nalini S. , Garen Alan , Kaplan Henry J.
Published: March 2003

Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. Severe loss of central vision frequently occurs with the exudative (wet) form of AMD, as a result of the formation of a pathological choroidal neovasculature (CNV) that damages the macular region of the retina. We tested the effect of an immunotherapy procedure, which had been shown to destroy the pathological neovasculature in solid tumors, on the formation of laser-induced CNV in a mouse model simulating exudative AMD in humans. The procedure involves administering an Icon molecule that binds with high affinity and specificity to tissue factor (TF), resulting in the activation of a potent cytolytic immune response against cells expressing TF. The Icon binds selectively to TF on the vascular endothelium of a CNV in the mouse and pig models and also on the CNV of patients with exudative AMD. Here we show that the Icon dramatically reduces the frequency of CNV formation in the mouse model. After laser treatment to induce CNV formation, the mice were injected either with an adenoviral vector encoding the Icon, resulting in synthesis of the Icon by vector-infected mouse cells, or with the Icon protein. The route of injection was i.v. or intraocular. The efficacy of the Icon in preventing formation of laser-induced CNV depends on binding selectively to the CNV. Because the Icon binds selectively to the CNV in exudative AMD as well as to laser-induced CNV, the Icon might also be efficacious for treating patients with exudative AMD.
Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism
Authors: Ma Ke , Cilingiroglu Mehmet , Otvos James D. , Ballantyne Christie M. , Marian Ali J. , Chan Lawrence
Published: March 2003

High-density lipoprotein (HDL) protects against atherosclerosis. Endothelial lipase (EL) has been postulated to be involved in lipoprotein, and possibly HDL, metabolism, yet the evidence has been scarce and conflicting. We have inactivated EL in mice by gene targeting. EL
−/−
mice have elevated plasma and HDL cholesterol, and increased apolipoproteins A-I and E. NMR analysis reveals an abundance of large HDL particles. There is down-regulation of the transcripts for phospholipid transfer protein, but up-regulation of those for hepatic lipase and lipoprotein lipase. Plasma lecithin:cholesterol acyltransferase is unchanged despite an increase in hepatic mRNA; lecithin:cholesterol acyltransferase activity toward endogenous EL
−/−
substrate is, however, reduced by 50%. HDL clearance is decreased in EL
−/−
mice; both the structure of HDL and the presence of EL are factors that determine the rate of clearance. To determine EL's role in humans, we find a significant association between a single-nucleotide polymorphism 584C/T in the EL (
LIPG
) gene and HDL cholesterol in a well characterized population of 372 individuals. We conclude that EL is a major determinant of HDL concentration, structure, and metabolism in mice, and a major determinant of HDL concentration in humans.
Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-κB site on its promoter
Authors: Simeonidis Simos , Castagliuolo Ignazio , Pan Amy , Liu Jennifer , Wang Chi-Chi , Mykoniatis Andreas , Pasha Asia , Valenick Leyla , Sougioultzis Stavros , Zhao Dezheng , Pothoulakis Charalabos
Published: March 2003

We report here that human monocytic/macrophage THP-1 cells express the neurokinin 1 receptor (NK-1R), and that exposure of these cells to the proinflammatory cytokine IL-1β increased the expression of the NK-1R gene at the mRNA and protein levels. Because IL-1β function involves nuclear factor κB (NF-κB) activation, these data suggest that this increase in the expression of the NK-1R gene is mediated by the NF-κB transcription factor. An earlier report noted that the promoter region of the human NK-1R gene contains a putative binding site for NF-κB [Takahashi, K., Tanaka, A., Hara, M. & Nakanishi, S. (1992)Eur. J. Biochem.204, 1025–1033]. Here we demonstrate that this is indeed a functional NF-κB-binding site, and that NF-κB is responsible for regulating the expression of the NK-1R gene by binding to the promoter region of the NK-1R gene. To further substantiate that the observed NF-κB-dependent IL-1β induction of the human NK-1R gene is regulated via a transcriptional event through this NF-κB site on the NK-1R gene promoter, we transfected THP-1 cells with a luciferase promoter-reporter construct containing the 5′ promoter region of the human NK-1R gene. Exposure of these cells to IL-1β or overexpression of NF-κB cDNAs resulted in a significant increase in the amount of luciferase activity that was diminished greatly in cells transfected with IκBα, the NF-κB inhibitor. These results directly implicate NF-κB in the regulation of the NK-1R gene and provide a molecular mechanism for the increase in expression of the NK-1R gene in responsive cells.
Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation
Authors: Ma Wen-ge , Song Haengseok , Das Sanjoy K. , Paria Bibhash C. , Dey Sudhansu K.
Published: March 2003

Many underlying causes of human infertility have been overcome by using
in vitro
fertilization (IVF) and embryo transfer (ET) techniques. Nevertheless, implantation rates in IVF programs remain low despite the transfer of apparently healthy embryos. This suggests that there are problems with the differentiation of the uterus to the receptive state in response to the ovarian hormones estrogen and progesterone. The molecular basis of this receptive state when the uterine environment is conducive to blastocyst acceptance and implantation remains poorly understood. Normally, the “window” of uterine receptivity lasts for a limited time. Using ETs and the progesterone-treated delayed-implantation model in mice, we demonstrate here that levels of estrogen within a very narrow range determine the duration of the window of uterine receptivity. Although estrogen at different physiological concentrations can initiate implantation, we find that the window of uterine receptivity remains open for an extended period at lower estrogen levels but rapidly closes at higher levels. The uterine refractoriness that follows the receptive state at high estrogen levels is accompanied by aberrant uterine expression of implantation-related genes. These results suggest that careful regulation of estrogen levels is one of the important factors for improvement of female fertility in IVF/ET programs.
Lake sediments record large-scale shifts in moisture regimes across the northern prairies of North America during the past two millennia
Authors: Laird Kathleen R. , Cumming Brian F. , Wunsam Sybille , Rusak James A. , Oglesby Robert J. , Fritz Sherilyn C. , Leavitt Peter R.
Published: March 2003

Six high-resolution climatic reconstructions, based on diatom analyses from lake sediment cores from the northern prairies of North America, show that shifts in drought conditions on decadal through multicentennial scales have prevailed in this region for at least the last two millennia. The predominant broad-scale pattern seen at all sites is a major shift in moisture regimes from wet to dry, or vice versa (depending on location), that occurred after a period of relative stability. These large-scale shifts at the different sites exhibit spatial coherence at regional scales. The three Canadian sites record this abrupt shift between
anno Domini
500 and 800, and subsequently conditions become increasingly variable. All three U.S. sites underwent a pronounced change, but the timing of this change is between
anno Domini
1000 and 1300, thus later than in all of the Canadian sites. The mechanisms behind these patterns are poorly understood, but they are likely related to changes in the shape and location of the jet stream and associated storm tracks. If the patterns seen at these sites are representative of the region, this observed pattern can have huge implications for future water availability in this region.
The p53-inducible TSAP6 gene product regulates apoptosis and the cell cycle and interacts with Nix and the Myt1 kinase
Authors: Passer Brent J. , Nancy-Portebois Vanessa , Amzallag Nathalie , Prieur Sylvie , Cans Christophe , Roborel de Climens Aude , Fiucci Giusy , Bouvard Véronique , Tuynder Marcel , Susini Laurent , Morchoisne Stéphanie , Crible Virginie , Lespagnol Alexandra , Dausset Jean , Oren Moshe , Amson Robert , Telerman Adam
Published: March 2003

The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST/
in vitro
-transcribed/translated pull-down assays and
in vivo
coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G
2
/M transition. Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.