bluuebunny/crossref_metadata_2025 huggingface dataset:
Ectopic expression of
N
-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid storage
Authors: Ihara Yoshito , Yoshimura Masafumi , Miyoshi Eiji , Nishikawa Atsushi , Sultan Ahmed S. , Toyosawa Satoru , Ohnishi Akio , Suzuki Misao , Yamamura Ken-ichi , Ijuhin Naokuni , Taniguchi Naoyuki
Published: July 2002

N
-Acetylglucosaminyltransferase III (GnT-III) produces “bisecting-GlcNAc” and regulates the branching of N-glycans. GnT-III activity is elevated during hepatocarcinogenesis, which is in contrast to the undetectable level found in normal hepatocytes. To determine the biological significance of GnT-III in hepatocytes, transgenic mice that specifically express GnT-III in the liver were established and characterized. The transgenic hepatocytes had a swollen oval-like morphology, with many lipid droplets. Apolipoprotein B, which contained increased level of bisecting-GlcNAc accumulated in the transgenic hepatocytes. In the transgenic serum, triglycerides, the β- and pre-β-lipoprotein fractions, and apolipoprotein B100 were significantly decreased, compared with levels in nontransgenic serum. These abnormal phenotypes were more prominent in the mice with more copies of the transgene and a resulting high GnT-III activity. We demonstrate that aberrant glycosylation, as the direct result of the formation of bisecting-GlcNAc, disrupts the function of apolipoprotein B, leading to the generation of fatty liver. This observation suggests a novel mechanism for the pathogenesis of fatty liver.
HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G
2
/M phase transition of the mammalian cell cycle
Authors: Mahalingam Sundarasamy , Ayyavoo Velpandi , Patel Mamata , Kieber-Emmons Thomas , Kao Gary D. , Muschel Ruth J. , Weiner David B.
Published: July 2002

Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr. These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G
2
/M phase border, immune suppression, and enhancement of virus replication. We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay. This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue. The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation. We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr
in vitro
and
in vivo
. hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade. We observe an association between the induction of cell cycle arrest at the G
2
/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization. This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity. We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.
Brownian dynamics simulations of protein folding: Access to milliseconds time scale and beyond
Authors: Rojnuckarin Atipat , Kim Sangtae , Subramaniam Shankar
Published: July 2002

Protein folding occurs on a time scale ranging from milliseconds to minutes for a majority of proteins. Computer simulation of protein folding, from a random configuration to the native structure, is nontrivial owing to the large disparity between the simulation and folding time scales. As an effort to overcome this limitation, simple models with idealized protein subdomains, e.g., the diffusion–collision model of Karplus and Weaver, have gained some popularity. We present here new results for the folding of a four-helix bundle within the framework of the diffusion–collision model. Even with such simplifying assumptions, a direct application of standard Brownian dynamics methods would consume 10,000 processor-years on current supercomputers. We circumvent this difficulty by invoking a special Brownian dynamics simulation. The method features the calculation of the mean passage time of an event from the flux overpopulation method and the sampling of events that lead to productive collisions even if their probability is extremely small (because of large free-energy barriers that separate them from the higher probability events). Using these developments, we demonstrate that a coarse-grained model of the four-helix bundle can be simulated in several days on current supercomputers. Furthermore, such simulations yield folding times that are in the range of time scales observed in experiments.
Recombination between diverged clusters of the tomato
Cf-9
plant disease resistance gene family
Authors: Parniske Martin , Jones Jonathan D. G.
Published: July 2002

The tomato
Cf-4
and
Cf-9
genes are the founder members of a large gene family of homologues of
Cladosporium fulvum
resistance gene
Cf-9
(
Hcr9
genes), several of which confer resistance against
C. fulvum
through recognition of different pathogen-encoded avirulence determinants. Three loci of tandemly repeated
Hcr9
genes—
Southern Cross
(
SC
),
Milky Way
(
MW
), and
Northern Lights
(
NL
)—are located on the short arm of tomato chromosome 1. Comparisons between 2
SC
-
Hcr9
s, 11 from
MW
, and 5 from
NL
implicated sequence exchange between gene family members in their evolution. The extent to which novel variants can be generated by recombination depends on the degree of sequence polymorphism available within the gene family. Here we show that physical separation of
Hcr9
genes can be associated with elevated sequence divergence. Two diverged subclasses of
Hcr9
s could be defined. These are physically separated from each other, with members of one class exclusively residing at
Northern Lights
. One exceptional
Hcr9
at
Northern Lights
carried sequence features specific for
Hcr9
s at other loci, suggesting a recent transfer of this gene by an interlocus recombination event. As members of diverged subclasses are brought into physical vicinity within a tandem repeat, a larger spectrum of sequence variants can potentially be generated by subsequent interhomologue sequence exchange.
Enhancer competition between
H19
and
Igf
2
does not mediate their imprinting
Authors: Schmidt Jennifer V. , Levorse John M. , Tilghman Shirley M.
Published: July 2002

The linked
H19
and
Igf2
genes on mouse distal chromosome 7 are subject to genomic imprinting. Competition between the promoters of the genes for transcription from shared enhancers has been proposed as an explanation for the coordinate expression and reciprocal imprinting of these two genes. To test this model, we have used Cre-loxP technology to generate in mice a conditional deletion of the
H19
promoter and structural gene that leaves no transcription unit in the locus. Contrary to the prediction of enhancer competition we find that transcriptional activity from the
H19
promoter is not required for the imprinted silencing of the
Igf2
gene.
Crystal structure of the DNA nucleotide excision repair enzyme UvrB from
Thermus thermophilus
Authors: Machius Mischa , Henry Lisa , Palnitkar Maya , Deisenhofer Johann
Published: July 2002

Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of
Thermus thermophilus
UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process.
Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation
Authors: Pereira de Araújo Antônio F.
Published: July 2002

The present study explores a “hydrophobic” energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its “hydrophobicity” and the number of contacts it makes, i.e.,
E(h⃗, c⃗) = −Σ

i=1

N

c
i
h
i
= −(h⃗.c⃗)

is the negative scalar product between two vectors in
N
-dimensional cartesian space:

h⃗ = (h
1
, … , h
N
)

, which represents monomer hydrophobicities and is sequence-dependent; and

c⃗ = (c
1
, … , c
N
)

, which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector
c⃗,
the best sequence is a vector
h⃗
on the direction upon which the projection of
c⃗ − c̄⃗
is maximal, where
c̄⃗
is the diagonal vector with components equal to
c̄,
the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.
Phosphorylation of presenilin-2 regulates its cleavage by caspases and retards progression of apoptosis
Authors: Walter Jochen , Schindzielorz Alice , Grünberg Jürgen , Haass Christian
Published: July 2002

Mutations within the Presenilin-2 (PS-2) gene are associated with
early onset familial Alzheimer’s disease. The gene encodes a polytopic
transmembrane protein that undergoes endoproteolytic processing
resulting in the generation of N-terminal and C-terminal fragments
(CTFs). PS-2 is also cleaved by proteases of the caspase family during
apoptotic cell death. CTFs of PS-2 were shown to inhibit
apoptosis, suggesting an important role in the regulation of
programmed cell death. Recently, we found that the CTF of PS-2 is
phosphorylated
in vivo
. We mapped the
in vivo
phosphorylation sites of PS-2 to serine residues 327 and 330, which are
localized immediately adjacent to the cleavage sites of caspases after
aspartate residues 326 and 329. Phosphorylation of PS-2 inhibits its
cleavage by caspase-3. This effect can be mimicked by substitutions of
serines 327 and 330 by aspartate or glutamate. In addition, the
uncleavable form of PS-2 CTF was found to enhance its
antiapoptotic properties, leading to a slower progression of
apoptosis. These results demonstrate that PS-2 cleavage as well
as its function in apoptosis can be regulated by protein
phosphorylation. Alterations in the phosphorylation of PS-2 may
therefore promote the pathogenesis of AD by affecting the
susceptibility of neurons to apoptotic stimuli.
Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces
Authors: Briesewitz Roger , Ray Gregory T. , Wandless Thomas J. , Crabtree Gerald R.
Published: July 2002

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.
In vitro
hematopoietic and endothelial potential of

flk-1
−/−

embryonic stem cells and embryos
Authors: Schuh Andre C. , Faloon Patrick , Hu Qing-Long , Bhimani Munsif , Choi Kyunghee
Published: July 2002

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die
in utero
because of defective vascular and hematopoietic development. Here, we show that
flk-1
−/−
embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells
in vitro
, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum
flk-1
−/−
embryos, even though 8.5-day postcoitum
flk-1
−/−
embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.
Replication fork assembly at recombination intermediates is required for bacterial growth
Authors: Liu Joing , Xu Liewei , Sandler Steven J. , Marians Kenneth J.
Published: July 2002

PriA, a 3′ → 5′ DNA helicase, directs assembly of a primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of
priA
null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the
priA2
∷
kan
mutation, bypasses the need for PriA during replication fork assembly at D loops
in vitro
. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.
Combinatorial protein engineering by incremental truncation
Authors: Ostermeier Marc , Nixon Andrew E. , Shim Jae Hoon , Benkovic Stephen J.
Published: July 2002

We have developed a combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on
Escherichia coli
glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.
Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis
Authors: Seo Naohiro , Tokura Yoshiki , Nishijima Takafumi , Hashizume Hideo , Furukawa Fukumi , Takigawa Masahiro
Published: July 2002

H-2Kb-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181–188 (TRP-2) and connexin 37 residues 52–59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Iab+cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.
The major transition state in folding need not involve the immobilization of side chains
Authors: Staniforth Rosemary A. , Dean Jonathan L. E. , Zhong Qi , Zerovnik Eva , Clarke Anthony R. , Waltho Jonathan P.
Published: July 2002

During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well.
Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse β-globin gene clusters
Authors: Bulger Michael , Bender M. A. , van Doorninck J. Hikke , Wertman Brett , Farrell Catherine M. , Felsenfeld Gary , Groudine Mark , Hardison Ross
Published: July 2002

By sequencing regions flanking the β-globin gene complex in mouse
(
Hbbc
) and human (
HBBC
), we have shown
that the β-globin gene cluster is surrounded by a larger cluster of
olfactory receptor genes (ORGs). To facilitate sequence comparisons and
to investigate the regulation of ORG expression, we have mapped 5′
sequences of mRNA from olfactory epithelium encoding
β-globin-proximal ORGs. We have found that several of these genes
contain multiple noncoding exons that can be alternatively spliced.
Surprisingly, the only common motifs found in the promoters of these
genes are a “TATA” box and a purine-rich motif. Sequence
comparisons between human and mouse reveal that most of the conserved
regions are confined to the coding regions and transcription units of
the genes themselves, but a few blocks of conserved sequence also are
found outside of ORG transcription units. The possible influence of
β-globin regulatory sequences on ORG expression in olfactory
epithelium was tested in mice containing a deletion of the
endogenous β-globin locus control region, but no change
in expression of the neighboring ORGs was detected. We evaluate the
implications of these results for possible mechanisms of regulation of
ORG transcription.
TNP‐470 Suppresses the Tumorigenicity of HT1080 Fibrosarcoma TumorThrough the Inhibition of VEGF Secretion From the Tumor Cells
Authors: Kaya Mitsunori , Wada Takuro , Nagoya Satoshi , Kawaguchi Satoshi , Yamashita Toshihiko , Yamamoto Nobuyuki , Yoshimoto Mitsunori , Okada Futoshi , Ishii Seiichi
Published: July 2002

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer. TNP‐470, a systemic analogue
of fumagillin, is an angiogenesis inhibitor capable of suppressing the tumorigenicity in several animal models even though
the mechanisms of action have not been completely clarified. In the current study, we investigated the effects of TNP‐470
on human fibrosarcoma cells in vivo and in vitro. The administration of TNP‐470 could suppress the tumorigenicity of
HT1080 fibrosarcoma tumor. The conditioned medium from HT1080 fibrosarcoma cells treated with TNP‐470 inhibited
the proliferation and migration of human endothelial cell line, HUVEC and ECV304. The concentration of VEGF in the
conditioned medium from HT1080 cells treated with TNP‐470 was lower than that of the cells without TNP‐470 treatment,
indicating that TNP‐470 downregulates the secretion of VEGF from HT1080 cells. These findings strongly suggest that the
direct action of TNP‐470 on sarcoma cells inhibits angiogenesis through the downregulation of VEGF secretion and this
angiogenesis suppression resulted in the inhibition of tumorigenicity of HT1080 fibrosarcoma tumo.
Imaging of Primitive Neuroectodermal Tumor (PNET) by Technetium‐99m Tetrofosmin
Authors: Karanikas Georgios , Köstler Wolfgang , Becherer Alexander , Dudczak Robert , Krainer Michael , Kletter Kurt
Published: July 2002

Purpose. Tc‐99m tetrofosmin launched for myocardial studies has recently also shown a good detectability for several tumors.
Data on PNET imaging by Tc‐99m tetrofosmin are not yet available.

Patient and method. In the case of a 21‐year‐old man suffering from pelvis PNET, Tc‐99m tetrofosmin scintigraphy was
performed additionally to CT and MRI.

Results. The gluteal and iliac tumor masses were visualized by Tc‐99m tetrofosmin according to the CT and MRI results.

Discussion. Imaging with Tc‐99m tetrofosmin could provide additional information to the available conventional radiological
imaging modalities for diagnosis of PNET, and could be a useful tool for the restaging of the primary tumor.
Role of ERK and p38 Mitogen‐Activated Protein Kinase Cascades in Gastric Mucosal Inflammatory Responses to Helicobacter pylori Lipopolysaccharide
Authors: Slomiany Bronislaw L. , Slomiany Amalia
Published: July 2002

The animal model of H. pylori lipopolysaccharide (LPS)‐induced gastritis was used to study the role of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) in the mucosal release of tumor necrosis factor‐ α (TNF‐ α) and endothelin‐1 (ET‐1) in response to H. pylori infection. Rats, pre‐treated with specific inhibitors of p38 and ERK pathways, SB203580 and PD98059, were submitted to intragastric application of H. pylori LPS and maintained on the daily regimen of the inhibitors for 4 days. In the absence of inhibitors, the LPS elicited a pattern of mucosal inflammatory responses resembling that of acute gastritis, and reflected in a massive increase in the mucosal level of ET‐1 and TNF‐ α. Administration of SB203580 led to a 63.4% reduction in the extent of inflammatory involvement, the level of ET‐1 fell by a 42% and TNF‐ α declined by a 52.3%, whereas PD98059 elicited a 21.2% reduction in the extent of inflammatory involvement and a 22.7% decrease in TNF‐ α, but had no effect on the LPS‐induced increase in ET‐1. A combination of both inhibitors, while exerting additive effect on TNF‐ α, produced no additional reduction in ET‐1 and the extent of inflammatory involvement achieved with SB203580 alone. The findings suggest that the p38 MAPK signaling pathway plays a key role in the mediation of gastric mucosal inflammatory reaction to H. pylori infection.
The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus
Authors: Huang Sui , Deerinck Thomas J. , Ellisman Mark H. , Spector David L.
Published: July 2002

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671–3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181–1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471–11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring ∼80–180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP–PTB fusion protein showed that at least three RRMs at either the COOH or NH2 terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.
In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast
Authors: Lechler Terry , Li Rong
Published: July 2002

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.
O-Glycosylation of Axl2/Bud10p by Pmt4p Is Required for Its Stability, Localization, and Function in Daughter Cells
Authors: Sanders Sylvia L. , Gentzsch Martina , Tanner Widmar , Herskowitz Ira
Published: July 2002

Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and α cells select axial sites; a/α cells utilize bipolar sites. Mutants specifically defective in axial budding were isolated from an α strain using pseudohyphal growth as an assay. We found that a and α mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not. PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M. Gentzsch, and W. Tanner. 1995. Yeast. 11:1345–1351). We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4. Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants. These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane.
Filamin Is Required for Ring Canal Assembly and Actin Organization during Drosophila Oogenesis
Authors: Li Min-gang , Serr Madeline , Edwards Kevin , Ludmann Susan , Yamamoto Daisuke , Tilney Lewis G. , Field Christine M. , Hays Thomas S.
Published: July 2002

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human α-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human α-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.
Matrix Gla Protein Is a Developmental Regulator of Chondrocyte Mineralization And, When Constitutively Expressed, Blocks Endochondral and Intramembranous Ossification in the Limb
Authors: Yagami Kimitoshi , Suh Jo-Young , Enomoto-Iwamoto Motomi , Koyama Eiki , Abrams William R. , Shapiro Irving M. , Pacifici Maurizio , Iwamoto Masahiro
Published: July 2002

Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.
Recycling of the Yeast a-Factor Receptor
Authors: Chen Linyi , Davis Nicholas G.
Published: July 2002

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.
Temporal and Spatial Distribution of Activated Pak1 in Fibroblasts
Authors: Sells Mary Ann , Pfaff Amanda , Chernoff Jonathan
Published: July 2002

p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.
In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles
Authors: Leung Anthony K.L. , Lamond Angus I.
Published: July 2002

The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization.
Role of Repetitive Antigen Patterns for Induction of Antibodies Against Antibodies
Authors: Fehr Thomas , Bachmann Martin F. , Bucher Etienne , Kalinke Ulrich , Padova Franco E. Di , Lang Alois B. , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis. Experience shows that they are usually difficult to induce experimentally. Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions. Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear. Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses. We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants. The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases.
Interleukin 12 and Interleukin 4 Control T Cell Adhesion to Endothelial Selectins through Opposite Effects on α1,3-fucosyltransferase VII Gene Expression
Authors: Wagers Amy J. , Waters Christopher M. , Stoolman Lloyd M. , Kansas Geoffrey S.
Published: July 2002

The α1,3-fucosyltransferase, FucT-VII, is crucial for the formation of ligands for all three selectins, and its expression regulates the synthesis of these ligands. Short-term polarized T helper (Th)1, but not Th2 or naive CD4+ T cells, can home to sites of inflammation, but the molecular basis for this difference has remained unclear. Here we show that naive CD4+ T cells do not express FucT-VII and fail to bind vascular selectins. We also show that when CD4+ T cells are activated in the presence of the Th1 polarizing cytokine interleukin (IL)-12, levels of FucT-VII mRNA and binding to E- and P-selectin are significantly augmented. In contrast, activation of CD4+ T cells in the presence of IL-4, a Th2 polarizing cytokine, inhibited FucT-VII expression and binding to vascular selectins. T cell activation upregulated expression of the Core2 transferase, C2GnT, equivalently regardless of the presence or absence of polarizing cytokines. These data indicate that the selective ability of Th1 cells, as opposed to Th2 cells or naive CD4+ T cells, to recognize vascular selectins and home to sites of inflammation is controlled principally by the expression of a single gene, FucT-VII.
Relaxed Negative Selection in Germinal Centers and Impaired Affinity Maturation in bcl-xL Transgenic Mice
Authors: Takahashi Yoshimasa , Cerasoli Douglas M. , Dal Porto Joseph M. , Shimoda Michiko , Freund Robert , Fang Wei , Telander David G. , Malvey Erika-Nell , Mueller Daniel L. , Behrens Timothy W. , Kelsoe Garnett
Published: July 2002

The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G1 antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.
Mapping the Energy of Superantigen Staphylococcus Enterotoxin C3 Recognition of an α/β T Cell Receptor Using Alanine Scanning Mutagenesis
Authors: Churchill Hywyn R.O. , Andersen Peter S. , Parke Evan A. , Mariuzza Roy A. , Kranz David M.
Published: July 2002

Binding of the T cell receptor (TCR) to a bacterial superantigen (SAG) results in stimulation of a large population of T cells and subsequent inflammatory reactions. To define the functional contribution of TCR residues to SAG recognition, binding by 24 single-site alanine substitutions in the TCR Vβ domain to Staphylococcus aureus enterotoxin (SE) C3 was measured, producing an energy map of the TCR–SAG interaction. The results showed that complementarity determining region 2 (CDR2) of the Vβ contributed the majority of binding energy, whereas hypervariable region 4 (HV4) and framework region 3 (FR3) contributed a minimal amount of energy. The crystal structure of the Vβ8.2–SEC3 complex suggests that the CDR2 mutations act by disrupting Vβ main chain interactions with SEC3, perhaps by affecting the conformation of CDR2. The finding that single Vβ side chain substitutions had significant effects on binding and that other SEC3-reactive Vβ are diverse at these same positions indicates that SEC3 binds to other TCRs through compensatory mechanisms. Thus, there appears to be strong selective pressure on SAGs to maintain binding to diverse T cells.
The Functional Binding Site for the C-Type Lectin–Like Natural Killer Cell Receptor Ly49a Spans Three Domains of Its Major Histocompatibility Complex Class I Ligand
Authors: Matsumoto Naoki , Mitsuki Motoaki , Tajima Kyoko , Yokoyama Wayne M. , Yamamoto Kazuo
Published: July 2002

Natural killer (NK) cells express receptors that recognize major histocompatibility complex (MHC) class I molecules and regulate cytotoxicity of target cells. In this study, we demonstrate that Ly49A, a prototypical C-type lectin–like receptor expressed on mouse NK cells, requires species-specific determinants on β2-microglobulin (β2m) to recognize its mouse MHC class I ligand, H-2Dd. The involvement of β2m in the interaction between Ly49A and H-2Dd is also demonstrated by the functional effects of a β2m-specific antibody. We also define three residues in α1/α2 and α3 domains of H-2Dd that are critical for the recognition of H-2Dd on target cells by Ly49A. In the crystal structure of the Ly49A/H-2Dd complex, these residues are involved in hydrogen bonding to Ly49A in one of the two potential Ly49A binding sites on H-2Dd. These data unambiguously indicate that the functional effect of Ly49A as an MHC class I–specific NK cell receptor is mediated by binding to a concave region formed by three structural domains of H-2Dd, which partially overlaps the CD8 binding site.
Adrenaline Stimulates Glucagon Secretion in Pancreatic A-Cells by Increasing the Ca2+ Current and the Number of Granules Close to the L-Type Ca2+ Channels
Authors: Gromada Jesper , Bokvist Krister , Ding Wei-Guang , Barg Sebastian , Buschard Karsten , Renström Erik , Rorsman Patrik
Published: July 2002

We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through ω-conotoxin-GVIA–sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via β-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.
Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases
Authors: Bowlby Mark R. , Fadool Debra A. , Holmes Todd C. , Levitan Irwin B.
Published: July 2002

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.