bluuebunny/crossref_metadata_2025 huggingface dataset:
Accuracy of lesion bypass by yeast and human DNA polymerase η
Authors: Washington M. Todd , Johnson Robert E. , Prakash Louise , Prakash Satya
Published: July 2002

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10
−2
to 10
−3
opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10
−2
to 10
−3
relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.
Presenilin and nicastrin regulate each other and determine amyloid β-peptide production via complex formation
Authors: Edbauer Dieter , Winkler Edith , Haass Christian , Steiner Harald
Published: July 2002

Amyloid β-peptide (Aβ) is generated by the consecutive cuts of two membrane-bound proteases. β-Secretase cuts at the N terminus of the Aβ domain, whereas γ-secretase mediates the C-terminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be γ-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active γ-secretases likely require other cofactors such as nicastrin (Nct) for their activities. Here we show that preferentially mature Nct forms a stable complex with PSs. Furthermore, we have down-regulated Nct levels by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the β-amyloid precursor protein. In addition, Aβ production was markedly reduced. Strikingly, down-regulation of Nct destabilized PS and strongly lowered levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in PS1
−/−
cells was associated with a strong down-regulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings we conclude that Nct and PS regulate each other and determine γ-secretase function via complex formation.
Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells
Authors: Pitari G. M. , Di Guglielmo M. D. , Park J. , Schulz S. , Waldman S. A.
Published: July 2002

The effects of
Escherichia coli
heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [
3
H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [
3
H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.
Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge
Authors: Chen Kevin C. , Nicholson Charles
Published: July 2002

Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor λ and volume fraction α. Recent studies in brain slices show that when osmolarity is reduced, λ increases while α decreases. In contrast, with increased osmolarity, α increases, but λ attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of λ can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference.
Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRα
Authors: Venkateswaran Asha , Laffitte Bryan A. , Joseph Sean B. , Mak Puiying A. , Wilpitz Damien C. , Edwards Peter A. , Tontonoz Peter
Published: July 2002

LXRα is a nuclear receptor that has previously been shown to
regulate the metabolic conversion of cholesterol to bile acids. Here we
define a role for this transcription factor in the control of cellular
cholesterol efflux. We demonstrate that retroviral expression of LXRα
in NIH 3T3 fibroblasts or RAW264.7 macrophages and/or treatment of
these cells with oxysterol ligands of LXR results in 7- to 30-fold
induction of the mRNA encoding the putative cholesterol/phospholipid
transporter ATP-binding cassette (ABC)A1. In contrast, induction of
ABCA1 mRNA in response to oxysterols is attenuated in cells that
constitutively express dominant-negative forms of LXRα or LXRβ that
lack the AF2 transcriptional activation domain. We further demonstrate
that expression of LXRα in NIH 3T3 fibroblasts and/or treatment of
these cells with oxysterols is sufficient to stimulate cholesterol
efflux to extracellular apolipoprotein AI. The ability of oxysterol
ligands of LXR to stimulate efflux is dramatically reduced in Tangier
fibroblasts, which carry a loss of function mutation in the ABCA1 gene.
Taken together, these results indicate that cellular cholesterol efflux
is controlled, at least in part, at the level of transcription by a
nuclear receptor–signaling pathway. They suggest a model in which
activation of LXRs by oxysterols in response to cellular sterol loading
leads to induction of the ABCA1 transporter and the stimulation of
lipid efflux to extracellular acceptors. These findings have important
implications for our understanding of mammalian cholesterol homeostasis
and suggest new opportunities for pharmacological regulation of
cellular lipid metabolism.
The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells
Authors: Morillon Raphaël , Chrispeels Maarten J.
Published: July 2002

The transpiration stream that passes through a plant may follow an apoplastic route, with low resistance to flow, or a cell-to-cell route, in which cellular membranes impede water flow. However, passage of water through membranes can be facilitated by aquaporins thereby decreasing resistance. We investigated the relationship between transpiration, which can be down-regulated by abscisic acid (ABA) or by high humidity, and the osmotic water permeability (
P

os

) of protoplasts. By using leaf protoplasts of wild-type (wt)
Arabidopsis thaliana
plants and of mutants that are low in ABA (
aba1
) or insensitive to ABA (
abi1
and
abi2
), we found that protoplasts from
aba1
and
abi
mutants have very low
P

os

values compared with those from wt plants when the plants are grown at 45% relative humidity. High values of
P

os

were found 3 h after the addition of ABA to the culture medium of
aba1
plants; addition of ABA to
abi
plants did not restore the
P

os

to wt levels. There was no such increase in
P

os

when excised leaves of
aba1
plants were treated with ABA. When the transpiration stream was attenuated by growing the plants at 85% relative humidity, the
P

os

of protoplasts from all plants (wt and mutants) was higher. We suggest that attenuation of the transpiration stream in whole plants is required for the up-regulation of the
P

os

of the membranes, and that this up-regulation, which does not require ABA, is mediated by the activation of aquaporins in the plasma membrane.
A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding
Authors: Harty Ronald N. , Brown Melissa E. , Wang Guangli , Huibregtse Jon , Hayes Felicia P.
Published: July 2002

VP40, the putative matrix protein of both Ebola and Marburg
viruses, possesses a conserved proline-rich motif (PY motif) at its N
terminus. We demonstrate that the VP40 protein can mediate its own
release from mammalian cells, and that the PY motif is important for
this self-exocytosis (budding) function. In addition, we used
Western-ligand blotting to demonstrate that the PY motif of VP40 can
mediate interactions with specific cellular proteins that have type I
WW-domains, including the mammalian ubiquitin ligase, Nedd4. Single
point mutations that disrupted the PY motif of VP40 abolished the
PY/WW-domain interactions. Significantly, the full-length VP40
protein was shown to interact both physically and functionally with
full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4. The
VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in
an
in vitro
ubiquitination assay. These data demonstrate
that the VP40 protein of Ebola virus possesses a PY motif that is
functionally similar to those described previously for Gag and M
proteins of specific retroviruses and rhabdoviruses, respectively.
Last, these studies imply that VP40 likely plays an important role in
filovirus budding, and that budding of retroviruses, rhabdoviruses, and
filoviruses may proceed via analogous mechanisms.
An autocatalytic mechanism of protein nitrosylation
Authors: Nedospasov Andrey , Rafikov Ruslan , Beda Natalya , Nudler Evgeny
Published: July 2002

Nitros(yl)ation is a widespread protein modification that occurs
during many physiological and pathological processes. It can alter both
the activity and function of a protein. Nitric oxide (
⋅
NO) has
been implicated in this process, but its mechanism remained uncertain.
⋅
NO is unable to react with nucleophiles under oxygen-free
conditions, suggesting that its higher oxides, such as
N
2
O
3
, were actually nitrosylating agents.
However, low concentrations and short lifespans of these species
in vivo
raise the question of how they could efficiently
locate target proteins. Here we demonstrate that at physiological
concentrations of
⋅
NO, N
2
O
3
forms inside
protein-hydrophobic cores and causes nitrosylation within the protein
interior. This mechanism of protein modification has not been
characterized, because all previously described mechanisms (e.g.,
phosphorylation, acetylation, ADP-ribosylation, etc.) occur via attack
on a protein by an external modification agent. Oxidation of
⋅
NO
to N
2
O
3
is facilitated by micellar catalysis,
which is mediated by the hydrophobic phase of proteins. Thus, a target
protein seems to be a catalyst of its own nitrosylation. One of the
applications of this finding, as we report here, is the design of
specific hydrophobic compounds whose cooperation with
⋅
NO and
O
2
allows the rapid inactivation of target enzymes to
occur.
Conservative front tracking and level set algorithms
Authors: Glimm James , Li Xiao Lin , Liu Yingjie , Zhao Ning
Published: July 2002

Hyperbolic conservation laws are foundational for many branches of
continuum physics. Discontinuities in the solutions of these partial
differential equations are widely recognized as a primary difficulty
for numerical simulation, especially for thermal and shear
discontinuities and fluid–fluid internal boundaries. We propose
numerical algorithms that will (
i
) track these
discontinuities as sharp internal boundaries, (
ii
) fully
conserve the conserved quantities at a discrete level, even at the
discontinuities, and (
iii
) display one order of numerical
accuracy higher globally (at the discontinuity) than algorithms in
common use. A significant improvement in simulation capabilities is
anticipated through use of the proposed algorithms.
Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.
Authors: Busch A E , Schuster A , Waldegger S , Wagner C A , Zempel G , Broer S , Biber J , Murer H , Lang F
Published: July 2002

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.
Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.
Authors: Jockusch S , Lee D , Turro N J , Leonard E F
Published: July 2002

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.
Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.
Authors: Knehr M , Poppe M , Schroeter D , Eickelbaum W , Finze E M , Kiesewetter U L , Enulescu M , Arand M , Paweletz N
Published: July 2002

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.
B cells are not essential for peripheral T-cell tolerance.
Authors: Vella A T , Scherer M T , Schultz L , Kappler J W , Marrack P
Published: July 2002

Some self-reactive T cells avoid thymic tolerance and become mature peripheral cells. Nevertheless, these cells do not usually attack their hosts because T cells can be inactivated or killed, even after they are mature, by various means. The details of these processes are not fully understood; however, a number of experiments have suggested that peripheral tolerance may be induced in mature mouse T cells by exposure to antigen on resting B cells, cells that can express antigen bound to major histocompatibility complex proteins but that lack critical costimulatory molecules such as B7-1 and B7-2. Conversely, previous experiments have indicated that mature T cells can be stimulated by exposure to antigen on cells such as dendritic cells, cells that are thought to express the essential costimulatory molecules. We tested this idea in vivo by using mice that lack B cells. Unexpectedly, T-cell tolerance and antigen-induced T-cell death occurred normally in mice free of B cells. On the other hand, antigen-specific T-cell expansion in the spleens of such mice was impaired. Finally, we have recently shown that T-cell death in mice can be prevented by exposure to antigen and an inflammatory agent such as bacterial lipopolysaccharide. This was also true in mice that lacked B cells. Overall, these data show that mature T cells can be tolerized and rescued from tolerance in the absence of B cells.
Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.
Authors: Issa J P , Vertino P M , Boehm C D , Newsham I F , Baylin S B
Published: July 2002

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.
Estrogen and thyroid hormone interaction on regulation of gene expression.
Authors: Zhu Y S , Yen P M , Chin W W , Pfaff D W
Published: July 2002

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.
National policies for technical change: Where are the increasing returns to economic research?
Authors: Pavitt Keith
Published: July 2002

Improvements over the past 30 years in statistical data, analysis, and related theory have strengthened the basis for science and technology policy by confirming the importance of technical change in national economic performance. But two important features of scientific and technological activities in the Organization for Economic Cooperation and Development countries are still not addressed adequately in mainstream economics: (i) the justification of public funding for basic research and (ii) persistent international differences in investment in research and development and related activities. In addition, one major gap is now emerging in our systems of empirical measurement—the development of software technology, especially in the service sector. There are therefore dangers of diminishing returns to the usefulness of economic research, which continues to rely completely on established theory and established statistical sources. Alternative propositions that deserve serious consideration are: (i) the economic usefulness of basic research is in the provision of (mainly tacit) skills rather than codified and applicable information; (ii) in developing and exploiting technological opportunities, institutional competencies are just as important as the incentive structures that they face; and (iii) software technology developed in traditional service sectors may now be a more important locus of technical change than software technology developed in “high-tech” manufacturing.
Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy
Authors: Oehlenschläger Frank , Schwille Petra , Eigen Manfred
Published: July 2002

Nucleic acid sequence-based amplification (NASBA) has proved to be
an ultrasensitive method for HIV-1 diagnosis in plasma even in the
primary HIV infection stage. This technique was combined with
fluorescence correlation spectroscopy (FCS) which enables online
detection of the HIV-1 RNA molecules amplified by NASBA. A
fluorescently labeled DNA probe at nanomolar concentration was
introduced into the NASBA reaction mixture and hybridizing to a
distinct sequence of the amplified RNA molecule. The specific
hybridization and extension of this probe during amplification
reaction, resulting in an increase of its diffusion time, was monitored
online by FCS. As a consequence, after having reached a critical
concentration of 0.1–1 nM (threshold for unaided FCS detection), the
number of amplified RNA molecules in the further course of reaction
could be determined. Evaluation of the hybridization/extension
kinetics allowed an estimation of the initial HIV-1 RNA concentration
that was present at the beginning of amplification. The value of
initial HIV-1 RNA number enables discrimination between positive and
false-positive samples (caused for instance by carryover
contamination)—this possibility of discrimination is an essential
necessity for all diagnostic methods using amplification systems (PCR
as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination
of NASBA with FCS may also be useful in assessing the efficacy of
anti-HIV agents, especially in the early infection stage when standard
ELISA antibody tests often display negative results.
Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun
Authors: Leonard David A. , Rajaram Nirmala , Kerppola Tom K.
Published: July 2002

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos–Jun–AP-1 complex may contribute to its functional versatility at different promoters.
An
in vitro
tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors
Authors: Sakurai Hiroyuki , Barros Elvino J. , Tsukamoto Tatsuo , Barasch Jonathan , Nigam Sanjay K.
Published: July 2002

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherin-positive, and ZO-1-positive by immunostaining) and BSN cells, which are largely mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an
in vitro
tubulogenesis system. BSN cells expressed hepatocyte growth factor (HGF) and transforming growth factor-β1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early
in vitro
branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor α, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens
in vitro
.
Role for Bcl-xLas an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis
Authors: Kharbanda Surender , Pandey Pramod , Schofield Lesley , Israels Sara , Roncinske Richard , Yoshida Kiyotsugu , Bharti Ajit , Yuan Zhi-Min , Saxena Satya , Weichselbaum Ralph , Nalin Carlo , Kufe Donald
Published: July 2002

Cytochrome C is a mitochondrial protein that induces apoptosis when released into the cytosol or when added to cell-free extracts. Here we show that cells that overexpress the Bcl-2-related protein Bcl-xLfail to accumulate cytosolic cytochrome C or undergo apoptosis in response to genotoxic stress. Coimmunoprecipitation studies demonstrate that Bcl-xLassociates with cytochrome C. Cytochrome C binds directly and specifically to Bcl-xLand not to the proapoptotic Bcl-xsprotein. The results also demonstrate that Bcl-xsblocks binding of cytochrome C to Bcl-xL. Our findings support a role for Bcl-xLin protecting cells from apoptosis by inhibiting the availability of cytochrome C in the cytosol.
Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1
Authors: Irie Atsushi , Kamata Tetsuji , Takada Yoshikazu
Published: July 2002

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997)Proc. Natl. Acad. Sci. USA94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.
A new class of obesity genes encodes leukocyte adhesion receptors
Authors: Dong Zhao Ming , Gutierrez-Ramos Jose-Carlos , Coxon Angela , Mayadas Tanya N. , Wagner Denisa D.
Published: July 2002

Obesity is a complex disease, and multiple genes contribute to the trait. The description of five genes (ob,db,tub,Ay, andfat) responsible for distinct syndromes of spontaneous monogenic obesity in mice has advanced our knowledge of the genetics of obesity. However, many other genes involved in the expression of this disease remain to be determined. We report here the identification of an additional class of genes involved in the regulation of adipose tissue mass. These genes encode receptors mediating leukocyte adhesion. Mice deficient in intercellular adhesion molecule-1 became spontaneously obese in old age on normal mouse chow or at a young age when provided with a diet rich in fat. Mice deficient in the counterreceptor for intercellular adhesion molecule-1, the leukocyte integrin αMβ2 (Mac-1), showed a similar obesity phenotype. Since all mice consumed approximately the same amount of food as controls, the leukocyte function appears to be in regulating lipid metabolism and/or energy expenditure. Our results indicate that (i) leukocytes play a role in preventing excess body fat deposition and (ii) defects in leukocyte adhesion receptors can result in obesity.
Potential roles of osteopontin and α
V
β
3
integrin in the development of coronary artery restenosis after angioplasty
Authors: Panda Dibyendu , Kundu Gopal C. , Lee Benjamin I. , Peri Alessandro , Fohl David , Chackalaparampil Isaac , Mukherjee Barid B. , Li Xiao D. , Mukherjee Diane C. , Seides Stuart , Rosenberg Joel , Stark Karen , Mukherjee Anil B.
Published: July 2002

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30–50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, α
v
β
3
integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with α
v
β
3
integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or α
v
β
3
integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-α
v
β
3
interaction was blocked by treatment of CASMCs with antibodies against OPN or α
v
β
3
integrin. Our results demonstrate that OPN and α
v
β
3
integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-α
v
β
3
interaction may provide a therapeutic approach to preventing restenosis.
Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: Up-regulation of poly(A) polymerase
Authors: Pendurthi Usha R. , Alok Deoraj , Rao L. Vijaya Mohan
Published: July 2002

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.
Function and regulation of heat shock factor 2 during mouse embryogenesis
Authors: Rallu M. , Loones Mt. , Lallemand Y. , Morimoto R. , Morange M. , Mezger V.
Published: July 2002

The spontaneous expression of heat shock genes during development is well documented in many animal species, but the mechanisms responsible for this developmental regulation are only poorly understood. In vertebrates, additional heat shock transcription factors, distinct from the heat shock factor 1 (HSF1) involved in the stress response, were suggested to be involved in this developmental control. In particular, the mouse HSF2 has been found to be active in testis and during preimplantation development. However, the role of HSF2 and its mechanism of activation have remained elusive due to the paucity of data on its expression during development. In this study, we have examined HSF2 expression during the postimplantation phase of mouse development. Our data show a developmental regulation of HSF2, which is expressed at least until 15.5 days of embryogenesis. It becomes restricted to the central nervous system during the second half of gestation. It is expressed in the ventricular layer of the neural tube which contains mitotically active cells but not in postmitotic neurons. Parallel results were obtained for mRNA, protein, and activity levels, demonstrating that the main level of control was transcriptional. The detailed analysis of the activity of a luciferase reporter gene under the control of the
hsp70.1
promoter, as well as the description of the protein expression patterns of the major heat shock proteins in the central nervous system, show that HSF2 and heat shock protein expression domains do not coincide. This result suggests that HFS2 might be involved in other regulatory developmental pathways and paves the way to new functional approaches.
Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein
Authors: Neufeld Kristi L. , White Raymond L.
Published: July 2002

Mutation of the adenomatous polyposis coli (
APC
) gene is an early step in the initiation of colon cancer. Because the distribution pattern of a protein within the cell can provide important clues as to function, we have used a combination of immunofluorescence microscopy and biochemical fractionation to determine the location of APC protein in epithelial cells. Immunofluorescence microscopy placed full-length APC protein in both the nucleus and the cytoplasm. The nuclear APC protein was concentrated in discrete subnuclear regions, including nucleoli, whereas the cytoplasmic APC protein concentrated at the leading edge of migrating cells. Colocalization of APC protein with rRNA confirmed a nucleolar localization. These immunocytochemical findings have been supported by cell fractionation, which demonstrated that full-length APC protein was located in both the membrane/cytoskeletal and the nuclear fractions.
Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts
Authors: Lee Ai Young , Akers Kathryn T. , Collier Mitchell , Li Lin , Eisen Arthur Z. , Seltzer Jo Louise
Published: July 2002

Normal fibroblasts cultured as monolayers secrete matrix metalloproteinases (MMP), including gelatinase A (72-kDa type IV collagenase) as inactive zymogens. Previously we found that normal fibroblasts cultured in a type I collagen lattice (dermal equivalent) secrete active gelatinase A. Here we show that the activation of progelatinase A occurs within the cell and that the activator copurifies with Golgi membranes. Cell extracts of fibroblasts cultured in collagen lattices contain active 62-kDa gelatinase A at least 4–6 h before active enzyme is detected in the culture medium. Pulse–chase experiments confirm these results. The activator is membrane-bound and localizes to the Golgi-enriched fraction. Highly purified plasma membranes from lattice cultures are unable to convert gelatinase A from the zymogen to its active form. The activator may be a metalloproteinase because EDTA prevents activation of exogenous proenzyme by membrane fractions. Membrane-type MMP1, the enzyme thought to be responsible for activation of gelatinase A on the plasma membrane of tumor cells, shows no significant change in either mRNA or protein levels during lattice culture. Intracellular levels of gelatinase A mRNA and protein increase during the culture period, and tissue inhibitor of metalloproteinases concentration does not change. Because of the greater availability of tissue inhibitor of metalloproteinases-free proenzyme as a substrate for the activator, it is possible that membrane-type MMP1 is the activating enzyme. In that case, malignant transformation may involve a change in the localization of the activator to the plasma membrane.
Molecular basis of plant-specific acid activation of K
+
uptake channels
Authors: Hoth Stefan , Dreyer Ingo , Dietrich Petra , Becker Dirk , Müller-Röber Bernd , Hedrich Rainer
Published: July 2002

During stomatal opening potassium uptake into guard cells and K
+
channel activation is tightly coupled to proton extrusion. The pH sensor of the K
+
uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K
+
channel in guard cell protoplasts from
Solanum tuberosum
(KST1), and the
kst1
gene product expressed in
Xenopus
oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K
+
channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K
+
uptake channels.
Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots
Authors: Kohli Ajay , Leech Mark , Vain Phillipe , Laurie David A. , Christou Paul
Published: July 2002

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through
Agrobacterium
, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of
Agrobacterium vir
genes in the integration process.
Variability in the stability of DNA–peptide nucleic acid (PNA) single-base mismatched duplexes: Real-time hybridization during affinity electrophoresis in PNA-containing gels
Authors: Igloi Gabor L.
Published: July 2002

The stability of all single-base mismatched pairs between a peptide nucleic acid 11-mer and its complementary DNA has been quantified in terms of their melting temperature and compared with the limited amount of data published to date. The strength of the interaction was determined by an automated affinity-electrophoretic approach permitting the visualization, in real time, of hybridization between a physically immobilized peptide nucleic acid and a complementary DNA migrating in an electric field. The dissociation constants are in the range of 10
−7
M (for mismatches) to 10
−10
M (for fully complementary DNA), which are in excellent agreement with solution studies. These and other thermodynamic constants can be accurately, rapidly, and reproducibly measured in this system at concentrations approaching dissociation conditions by using fluorescently labeled DNA in conjunction with commercial DNA sequencers. The stability of single-base mismatched peptide nucleic acid–DNA duplexes depends both on the position as well as on the chemical nature of the mismatch. The stability is at a minimum when the mutation is positioned 4 bases from either terminus (a loss of 20°C or more in the melting temperature) but regains substantial stability when the mismatch is at the center of the duplex. The most stable mismatched pairs are G:T and T:T whereas destabilization is maximal for A:A and G:G. These observations are of significance in the design of probes for detecting mutations by hybridization.