bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells
Authors: Kanazawa Takashi , Watanabe Masahiko , Matsushima-Hibiya Yuko , Kono Takuo , Tanaka Noriaki , Koyama Kotaro , Sugimura Takashi , Wakabayashi Keiji
Published: July 2002

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage
butterfly,
Pieris rapae
, and has been suggested to
consist of an N-terminal region with ADP-ribosyltransferase domain and
of a C-terminal region that might have a receptor-binding domain. To
elucidate the role of each region, we investigated the functions of
various fragments of pierisin-1.
In vitro
expressed
polypeptide consisting of amino acid residues 1–233 or 234–850 of
pierisin-1 alone did not show cytotoxicity against human cervical
carcinoma HeLa cells. However, the presence of both polypeptides in the
culture medium showed some of the original cytotoxic activity.
Introduction of the N-terminal polypeptide alone by electroporation
also induced cell death in HeLa cells, and even in the mouse melanoma
MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a
principal role in the cytotoxicity of pierisin-1 inside mammalian
cells. Analyses of incorporated pierisin-1 indicated that the entire
protein, regardless of whether it consisted of a single polypeptide or
two separate N- and C-terminal polypeptides, was incorporated into HeLa
cells. However, neither of the terminal polypeptides was incorporated
when each polypeptide was present separately. These findings indicate
that the C-terminal region is important for the incorporation of
pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid
fraction of cell membrane was suggested. The cytotoxic effects of
pierisin-1 were enhanced by previous treatment with trypsin, producing
“nicked” pierisin-1. Generation of the N-terminal fragment in
HeLa cells was detected after application of intact entire molecule of
pierisin-1. From the above observations, it is suggested that after
incorporation of pierisin-1 into the cell by interaction of its
C-terminal region with the receptor in the cell membrane, the entire
protein is cleaved into the N- and C-terminal fragments with
intracellular protease, and the N-terminal fragment then exhibits
cytotoxicity.
Enhanced signaling through the IL-2 receptor in CD8+T cells regulated by antigen recognition results in preferential proliferation and expansion of responding CD8+T cells rather than promotion of cell death
Authors: Cheng Laurence E. , Öhlén Claes , Nelson Brad H. , Greenberg Philip D.
Published: July 2002

Multiple cytokines, including IL-2, can affect T cell proliferation and survival. However, IL-2 can lead to apoptosis as well as proliferation, making unclear whether IL-2 receptor (IL-2R) signals ultimately have a predominantly positive or negative effect. To address this issue, we examined the effect of enhancing IL-2R signals in CD8+T cells after antigen stimulation by engineering a transgenic (Tg) mouse strain with CD8+T cells capable of augmented, regulated, autocrine IL-2R signaling after target recognition by means of expression of a chimeric granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-2R. The Tg CD8+T cells can bind the granulocyte-macrophage colony-stimulating GM-CSF produced by antigen stimulation, but the GM-CSF binding results in delivery of an IL-2R signal. After antigen stimulationin vivo, the Tg T cells demonstrated marked increases in the initial proliferative response and cell expansion and displayed continued increases in cell expansion after repeated antigen exposure. These data suggest that the predominant role of IL-2R signals delivered to responding CD8+T cells is to set the size of the initial response to antigen by promoting T cell proliferation and survival and not cell death.
2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB
1
receptor
Authors: Hanuš Lumír , Abu-Lafi Saleh , Fride Ester , Breuer Aviva , Vogel Zvi , Shalev Deborah E. , Kustanovich Irina , Mechoulam Raphael
Published: July 2002

Two types of endogenous cannabinoid-receptor agonists
have been identified thus far. They are the ethanolamides of
polyunsaturated fatty acids—arachidonoyl ethanolamide (anandamide) is
the best known compound in the amide series—and 2-arachidonoyl
glycerol, the only known endocannabinoid in the ester series. We report
now an example of a third, ether-type endocannabinoid, 2-arachidonyl
glyceryl ether (noladin ether), isolated from porcine brain. The
structure of noladin ether was determined by mass spectrometry and
nuclear magnetic resonance spectroscopy and was confirmed by comparison
with a synthetic sample. It binds to the CB
1
cannabinoid
receptor (
K
i
= 21.2 ± 0.5 nM) and
causes sedation, hypothermia, intestinal immobility, and mild
antinociception in mice. It binds weakly to the CB
2
receptor (
K
i
> 3 μM).
A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of α2
Authors: Smith Rebecca L. , Johnson Alexander D.
Published: July 2002

The tetratricopeptide repeat (TPR) is a 34-aa sequence motif, typically found in tandem clusters, that occurs in proteins of bacteria, archea, and eukaryotes. TPRs interact with other proteins, although few details on TPR–protein interactions are known. In this paper we show that a portion of a loop in the homeodomain of the DNA-binding protein α2 is required for its recognition by the TPRs of the corepressor Ssn6. The amino acid sequence of this loop is similar to the sequences recognized by the TPRs of an entirely different protein, Pex5, which directs peroxisomal import. We further show that α2 can be made to bind specifically
in vitro
to the TPRs of Pex5 and that a point mutation that disrupts the α2-Ssn6 interaction also disrupts the α2-Pex5 interaction. These results demonstrate that two different TPR proteins recognize their target by a similar mechanism, raising the possibility that other TPR-target interactions could occur through the same means.
Global biodiversity and the ancient carbon cycle
Authors: Rothman Daniel H.
Published: July 2002

Paleontological data for the diversity of marine animals and
land plants are shown to correlate significantly with a concurrent
measure of stable carbon isotope fractionation for approximately the
last 400 million years. The correlations can be deduced from the
assumption that increasing plant diversity led to increasing chemical
weathering of rocks and therefore an increasing flux of carbon from the
atmosphere to rocks, and nutrients from the continents to the oceans.
The CO
2
concentration dependence of photosynthetic carbon
isotope fractionation then indicates that the diversification of land
plants led to decreasing CO
2
levels, while the
diversification of marine animals derived from increasing nutrient
availability. Under the explicit assumption that global biodiversity
grows with global biomass, the conservation of carbon shows that the
long-term fluctuations of CO
2
levels were dominated by
complementary changes in the biological and fluid reservoirs of carbon,
while the much larger geological reservoir remained relatively constant
in size. As a consequence, the paleontological record of biodiversity
provides an indirect estimate of the fluctuations of ancient
CO
2
levels.
Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes
Authors: Kicska Greg A. , Long Li , Hörig Heidi , Fairchild Craig , Tyler Peter C. , Furneaux Richard H. , Schramm Vern L. , Kaufman Howard L.
Published: July 2002

Transition-state theory has led to the design of Immucillin-H
(Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase
(PNP). In humans, PNP is the only route for degradation of
deoxyguanosine, and genetic deficiency of this enzyme leads to
profound T cell-mediated immunosuppression. This study reports the
biological effects and mechanism of action of Imm-H on malignant T cell
lines and on normal activated human peripheral T cells. Imm-H inhibits
the growth of malignant T cell leukemia lines with the induction of
apoptosis. Imm-H also inhibits activated normal human T cells
after antigenic stimulation
in vitro
. However, Imm-H did
not inhibit malignant B cells, colon cancer cell lines, or normal human
nonstimulated T cells, demonstrating the selective activity of Imm-H.
The effects on leukemia cells were mediated by the cellular
phosphorylation of deoxyguanosine and the accumulation of dGTP, an
inhibitor of ribonucleotide diphosphate reductase. Cells were protected
from the toxic effects of Imm-H when deoxyguanosine was absent or
when deoxycytidine was present. Guanosine incorporation into nucleic
acids was selectively blocked by Imm-H with no effect on guanine,
adenine, adenosine, or deoxycytidine incorporation. Imm-H may have
clinical potential for treatment of human T cell leukemia and lymphoma
and for other diseases characterized by abnormal activation of T
lymphocytes. The design of Imm-H from an enzymatic transition-state
analysis exemplifies a powerful approach for developing high-affinity
enzyme inhibitors with pharmacologic activity.
Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2Kdthat stimulates CD8 T cells in insulin-dependent diabetes mellitus
Authors: Wong F. Susan , Moustakas Antonis K. , Wen Li , Papadopoulos George K. , Janeway Charles A.
Published: July 2002

The recognition of MHC–peptide complexes by T cells is governed by structural considerations that are determined by the sequences of the individual components and their interaction with each other. We have studied the function of a highly diabetogenic CD8 T cell clone that is specific for insulin B15-23:H-2Kd. We have then related this to modeled MHC–peptide structures. The native peptide binds poorly to H-2Kd, because of the small glycine residue at peptide position p9 that is incapable of productive interactions with the hydrophobic residues of pocket F. In addition, electrostatic repulsions between the peptide glutamate residue at position 7 and 152D of the MHC molecule heavy chain contribute to the poor binding. However, B chain peptide 15-23 bound to Kdshows excellent T cell stimulation and the induction of CD8 cytotoxic T cells. Peptide substitution has also shown that p6G is likely to be a T cell antigen receptor interaction site. Our studies have shown that the predictions seen in the models correlate closely with the observed effects in functional assays and provide insight into how this peptide, which would not be predicted to stimulate these cells on H-2Kdbinding studies alone, could activate such highly pathogenic T cells.
Aldosterone signaling pathway across the nuclear envelope
Authors: Schäfer C. , Shahin V. , Albermann L. , Hug M. J. , Reinhardt J. , Schillers H. , Schneider S. W. , Oberleithner H.
Published: July 2002

We describe the route by which aldosterone-triggered macromolecules enter and exit the cell nucleus of
Xenopus
laevis
oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2–30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5–15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small (≅100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large (≅800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (
i
) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (
ii
) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (
iii
) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.
The crystal structure of the C-terminal fragment of striated-muscle α-tropomyosin reveals a key troponin T recognition site
Authors: Li Yu , Mui Suet , Brown Jerry H. , Strand James , Reshetnikova Ludmilla , Tobacman Larry S. , Cohen Carolyn
Published: July 2002

Contraction in striated and cardiac muscles is regulated by the motions of a Ca
2+
-sensitive tropomyosin/troponin switch. In contrast, troponin is absent in other muscle types and in nonmuscle cells, and actomyosin regulation is myosin-linked. Here we report an unusual crystal structure at 2.7 Å of the C-terminal 31 residues of rat striated-muscle α-tropomyosin (preceded by a fragment of the GCN4 leucine zipper). The C-terminal 22 residues (263–284) of the structure do not form a two-stranded α-helical coiled coil as does the rest of the molecule, but here the α-helices splay apart and are stabilized by the formation of a tail-to-tail dimer with a symmetry-related molecule. The site of splaying involves a small group of destabilizing core residues that is present only in striated muscle tropomyosin isoforms. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles.
Stochastic focusing: Fluctuation-enhanced sensitivity of intracellular regulation
Authors: Paulsson Johan , Berg Otto G. , Ehrenberg Måns
Published: July 2002

Many regulatory molecules are present in low copy numbers per cell so that significant random fluctuations emerge spontaneously. Because cell viability depends on precise regulation of key events, such signal noise has been thought to impose a threat that cells must carefully eliminate. However, the precision of control is also greatly affected by the regulatory mechanisms' capacity for sensitivity amplification. Here we show that even if signal noise reduces the capacity for sensitivity amplification of threshold mechanisms, the effect on realistic regulatory kinetics can be the opposite: stochastic focusing (SF). SF particularly exploits tails of probability distributions and can be formulated as conventional multistep sensitivity amplification where signal noise provides the degrees of freedom. When signal fluctuations are sufficiently rapid, effects of time correlations in signal-dependent rates are negligible and SF works just like conventional sensitivity amplification. This means that, quite counterintuitively, signal noise can reduce the uncertainty in regulated processes. SF is exemplified by standard hyperbolic inhibition, and all probability distributions for signal noise are first derived from underlying chemical master equations. The negative binomial is suggested as a paradigmatic distribution for intracellular kinetics, applicable to stochastic gene expression as well as simple systems with Michaelis–Menten degradation or positive feedback. SF resembles stochastic resonance in that noise facilitates signal detection in nonlinear systems, but stochastic resonance is related to how noise in threshold systems allows for detection of subthreshold signals and SF describes how fluctuations can make a gradual response mechanism work more like a threshold mechanism.
Multiple differences in gene expression in regulatory Vα24JαQ T cells from identical twins discordant for type I diabetes
Authors: Wilson S. Brian , Kent Sally C. , Horton Heidi F. , Hill Andrew A. , Bollyky Paul L. , Hafler David A. , Strominger Jack L. , Byrne Michael C.
Published: July 2002

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Vα24JαQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Vα24JαQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.
Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of
Trypanosoma cruzi
Authors: Machado Carlos A. , Ayala Francisco J.
Published: July 2002

Simple phylogenetic tests were applied to a large data set of nucleotide sequences from two nuclear genes and a region of the mitochondrial genome of
Trypanosoma cruzi
, the agent of Chagas' disease. Incongruent gene genealogies manifest genetic exchange among distantly related lineages of
T. cruzi
. Two widely distributed isoenzyme types of
T. cruzi
are hybrids, their genetic composition being the likely result of genetic exchange between two distantly related lineages. The data show that the reference strain for the
T. cruzi
genome project (CL Brener) is a hybrid. Well-supported gene genealogies show that mitochondrial and nuclear gene sequences from
T. cruzi
cluster, respectively, in three or four distinct clades that do not fully correspond to the two previously defined major lineages of
T. cruzi
. There is clear genetic differentiation among the major groups of sequences, but genetic diversity within each major group is low. We estimate that the major extant lineages of
T. cruzi
have diverged during the Miocene or early Pliocene (3–16 million years ago).
A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53
Authors: Noolandi J. , Davison T. S. , Völkel A. R. , Nie X.-F. , Kay C. , Arrowsmith C. H.
Published: July 2002

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer–dimer interface of the tetrameric oligomerization domain of p53 (residues 325–355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer–dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.
A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage
Authors: Vispé Stéphane , Yung Tetsu M. C. , Ritchot Janelle , Serizawa Hiroaki , Satoh Masahiko S.
Published: July 2002

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD
+
, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.
Ataxia telangiectasia-mutated phosphorylates Chk2
in vivo
and
in vitro
Authors: Matsuoka Shuhei , Rotman Galit , Ogawa Akira , Shiloh Yosef , Tamai Katsuyuki , Elledge Stephen J.
Published: July 2002

The protein kinase Chk2, the mammalian homolog of the budding yeast
Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and
activated in response to DNA damage by ionizing radiation (IR), UV
irradiation, and replication blocks by hydroxyurea (HU).
Phosphorylation and activation of Chk2 are ataxia
telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2
phosphorylation is ATM-independent when cells are exposed to UV or HU.
Here we show that
in vitro
, ATM phosphorylates the
Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which
contains seven SQ/TQ motifs, and Thr68 is the major
in
vitro
phosphorylation site by ATM. ATM- and Rad3-related also
phosphorylates Thr68 in addition to Thr26 and Ser50, which are not
phosphorylated to a significant extent by ATM
in vitro
.
In vivo
, Thr68 is phosphorylated in an ATM-dependent
manner in response to IR, but not in response to UV or HU. Substitution
of Thr68 with Ala reduced the extent of phosphorylation and activation
of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs
blocked all phosphorylation and activation of Chk2 after IR. These
results suggest that
in vivo
, Chk2 is directly
phosphorylated by ATM in response to IR and that Chk2 is regulated by
phosphorylation of the SCD.
Spontaneous tumorigenesis in mice defective in the
MTH1
gene encoding 8-oxo-dGTPase
Authors: Tsuzuki Teruhisa , Egashira Akinori , Igarashi Hisato , Iwakuma Tomoo , Nakatsuru Yoko , Tominaga Yohei , Kawate Hisaya , Nakao Kazuki , Nakamura Kenji , Ide Fumio , Kura Shinobu , Nakabeppu Yusaku , Katsuki Motoya , Ishikawa Takatoshi , Sekiguchi Mutsuo
Published: July 2002

Oxygen radicals, which can be produced through normal cellular
metabolism, are thought to play an important role in mutagenesis and
tumorigenesis. Among various classes of oxidative DNA damage,
8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its
abundance and mutagenicity. The
MTH1
gene encodes an
enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide
pool, thereby preventing occurrence of transversion mutations. By means
of gene targeting, we have established
MTH1
gene-knockout cell lines and mice. When examined 18 months after birth,
a greater number of tumors were formed in the lungs, livers, and
stomachs of
MTH1
-deficient mice, as compared with
wild-type mice. The
MTH1
-deficient mouse will provide a
useful model for investigating the role of the MTH1 protein in normal
conditions and under oxidative stress.
Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a
Reb
-responsive promoter
Authors: Yang Daichang , Wu Liying , Hwang Yong-Sic , Chen Lifang , Huang Ning
Published: July 2002

The gene encoding the rice transcription factor, REB
(
r
ice
e
ndosperm
b
ZIP) was cloned
from a bacterial artificial chromosome library of rice. The cloned
6,227-bp-long
Reb
gene is composed of six exons and five
introns and is flanked by a 1.2-kb 5′ promoter and a 1.2-kb 3′
terminator region. The function of the
Reb
gene was
explored by a transient assay by using a rice immature endosperm
system. The effector constructs containing the native gene or fusion
genes linking
Reb
to the rice actin (
Act
)
or globulin (
Glb
) gene promoters and the reporter gene
construct Glb-β-glucuronidase (GUS) were used in this study. When
these effector constructs were cotransferred with the reporter
uid
A gene encoding GUS under the control of the
Glb
promoter into immature rice endosperm cells, the
Glb
promoter was activated. The transient GUS expression
was 2.0 to 2.5-fold higher with the effector construct than without.
When the upstream activation sequence containing the GCCACGT(A/C)AG
motifs of the
Glb
promoter was deleted, the activation
by REB was abolished. On the other hand, a gain-of-function experiment
showed that inserting the upstream activation sequence into the
glutelin-1 (
Gt1
) promoter made it responsive to
activation by REB. When cotransformed with
Reb
gene,
mature transgenic rice grains containing the human lysozyme gene driven
by the
Glb
promoter produced 3.7-fold more lysozyme.
Accumulation of recombinant lysozyme in mature seed ranged from 30.57
to 279.61 μg⋅mg
−1
total soluble protein in
individual transformants from 30 independent transformation events.
Thus, our results show that REB is not only a transcriptional
activator, it can also be used to increase the expression of
recombinant protein in transgenic rice grains.
Beryllium presentation to CD4+T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease
Authors: Fontenot Andrew P. , Torres Michaelann , Marshall William H. , Newman Lee S. , Kotzin Brian L.
Published: July 2002

Chronic beryllium disease results from beryllium exposure in the workplace and is characterized by CD4+T cell-mediated inflammation in the lung. Susceptibility to this disease is associated with particular HLA-DP alleles. We isolated beryllium-specific T cell lines from the lungs of affected patients. These CD4+T cell lines specifically responded to beryllium in culture in the presence of antigen-presenting cells that expressed class II MHC molecules HLA-DR, -DQ, and -DP. The response to beryllium was nearly completely and selectively blocked by mAb to HLA-DP. Additional studies showed that only certain HLA-DP alleles allowed presentation of beryllium. Overall, the DP alleles that presented beryllium to disease-specific T cell lines match those implicated in disease susceptibility, providing a mechanism for this association. Based on amino acid residues shared by these restricting and susceptibility DP alleles, our results provide insight into the residues of the DP β-chain required for beryllium presentation.
Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity
Authors: Rousso Itay , Mixon Mark B. , Chen Benjamin K. , Kim Peter S.
Published: July 2002

Recent studies suggest that HIV-1 budding occurs selectively from
detergent-insoluble membrane domains, referred to as lipid rafts.
Palmitoylation is thought to be one of the factors responsible for
targeting membrane proteins to lipid rafts. The cytoplasmic domain of
the HIV-1 envelope glycoprotein (gp160) contains two palmitoylated
cysteine residues. In this work, we studied the solubility of gp160
after detergent extraction. We show that wild-type gp160 is mostly
insoluble after ice-cold Triton X-100 extraction, but that it becomes
almost completely soluble at 37°C. In contrast, we find that a mutant
gp160, in which the two palmitoylated cysteine residues are replaced by
serine, is Triton X-100 soluble even under ice-cold extraction. These
findings are consistent with the properties of proteins that localize
to lipid rafts and strongly suggest that gp160 is associated with lipid
rafts. Further, removal of both palmitoylation sites results in the
formation of virus with low levels of gp160 incorporation as well as a
decrease in viral infectivity by 60-fold. Our results strongly support
the suggestion that HIV-1 buds from lipid rafts and point to a role for
rafts as a viral assembly hub.
Hotspots and the conservation of evolutionary history
Authors: Sechrest Wes , Brooks Thomas M. , da Fonseca Gustavo A. B. , Konstant William R. , Mittermeier Russell A. , Purvis Andy , Rylands Anthony B. , Gittleman John L.
Published: July 2002

Species diversity is unevenly distributed across the globe, with terrestrial diversity concentrated in a few restricted biodiversity hotspots. These areas are associated with high losses of primary vegetation and increased human population density, resulting in growing numbers of threatened species. We show that conservation of these hotspots is critical because they harbor even greater amounts of evolutionary history than expected by species numbers alone. We used supertrees for carnivores and primates to estimate that nearly 70% of the total amount of evolutionary history represented in these groups is found in 25 biodiversity hotspots.
Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation.
Authors: Melo-Oliveira R , Oliveira I C , Coruzzi G M
Published: July 2002

Glutamate dehydrogenase (GDH) is ubiquitous to all organisms, yet its role in higher plants remains enigmatic. To better understand the role of GDH in plant nitrogen metabolism, we have characterized an Arabidopsis mutant (gdh1-1) defective in one of two GDH gene products and have studied GDH1 gene expression. GDH1 mRNA accumulates to highest levels in dark-adapted or sucrose-starved plants, and light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The Arabidopsis gdh-deficient mutant allele gdh1-1 cosegregates with the GDH1 gene and behaves as a recessive mutation. The gdh1-1 mutant displays a conditional phenotype in that seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen. These results suggest that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess. This notion is further supported by the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.
Age-specific inbreeding depression and components of genetic variance in relation to the evolution of senescence.
Authors: Charlesworth B , Hughes K A
Published: July 2002

Two major theories of the evolution of senescence (mutation accumulation and antagonistic pleiotropy) make different predictions about the relationships between age, inbreeding effects, and the magnitude of genetic variance components of life-history components. We show that, under mutation accumulation, inbreeding decline and three major components of genetic variance are expected to increase with age in randomly mating populations. Under the simplest version of the antagonistic pleiotropy model, no changes in the severity of inbreeding decline, dominance variance, or the genetic variance of chromosomal homozygotes are expected, but additive genetic variance may increase with age. Age-specific survival rates and mating success were measured on virgin males, using lines extracted from a population of Drosophila melanogaster. For both traits, inbreeding decline and several components of genetic variance increase with age. The results are consistent with the mutation accumulation model, but can only be explained by antagonistic pleiotropy if there is a general tendency for an increase with age in the size of allelic effects on these life-history traits.
Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.
Authors: Lovett S T , Feschenko V V
Published: July 2002

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.
Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.
Authors: Kitamura M
Published: July 2002

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.
Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.
Authors: Lütjohann D , Breuer O , Ahlborg G , Nennesmo I , Sidén A , Diczfalusy U , Björkhem I
Published: July 2002

We have investigated whether side chain-hydroxylated cholesterol species are important for elimination of cholesterol from the brain. Plasma concentrations of 24-hydroxycholesterol (24-OH-Chol) in the internal jugular vein and the brachial artery in healthy volunteers were consistent with a net flux of this steroid from the brain into the circulation, corresponding to elimination of approximately 4 mg cholesterol during a 24-h period in adults. Results of experiments with rats exposed to 18O2 were also consistent with a flux of 24-OH-Chol from the brain into the circulation. No other oxysterol measured showed a similar behavior as 24-OH-Chol. These results and the finding that the concentration of 24-OH-Chol was 30- to 1500-fold higher in the brain than in any other organ except the adrenals indicate that the major part of 24-OH-Chol present in the circulation originates from the brain. Both the 24-OH-Chol present in the brain and in the circulation were the 24S-stereoisomer. In contrast to other oxysterols, levels of plasma 24-OH-Chol were found to be markedly dependent upon age. The ratio between 24-OH-Chol and cholesterol in plasma was approximately 5 times higher during the first decade of life than during the sixth decade. There was a high correlation between levels of 24-OH-Chol in plasma and cerebrospinal fluid. It is suggested that the flux of 24-OH-Chol from the brain is important for cholesterol homeostasis in this organ.
Influence of light on DNA content of Helianthus annuus Linnaeus.
Authors: Price H J , Johnston J S
Published: July 2002

Mean nuclear 2C DNA content (C equaling haploid DNA per nucleus) of the first leaf of the sunflower, Helianthus annuus L., is influenced by the quality and the quantity of light. Seedlings of two inbred lines, RHA 299 and RHA 271 were germinated and grown in controlled environmental conditions. Lighting was adjusted to provide different combinations of photon flux densities and red to far red (R:FR) ratios. At R:FR = 5.8 and photon flux densities of 170 mumol.m-2.s-1, 200 mumol.m-2.s-1, and 230 mumol.m-2.s-1, DNA content remained high and relatively constant (x = 6.97 pg for RHA 271 and x = 7.32 pg for RHA 299). When the photon flux density range (R:FR = 5.8) was elevated to 350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1, mean DNA content was reduced to 6.23 pg (RHA 271) and 6.46 pg (RHA 299). At R:FR = 1.5, mean DNA content was consistently high (7.2-7.9 pg) only at the lowest photon flux density of 170 mumol.m-2.s-1. Significant decreases in DNA content (< or = 12%) were observed at photon flux densities of 200 mumol.m-2.s-1 and 230 mumol.m-2.s-1. At the higher photon flux densities (350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1) and R:RF = 1.5, the plants had extremely low DNA contents (mean x = 3.36 pg for RHA 271 and 3.41 pg for RHA 299) and high between-plant variance. The instability of DNA content, particularly for plants grown under light that is far red rich, suggests that phytochromes may be involved in regulating DNA content of the sunflower.
Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice
Authors: Witzemann V. , Schwarz H. , Koenen M. , Berberich C. , Villarroel A. , Wernig A. , Brenner H. R. , Sakmann B.
Published: July 2002

In mammalian muscle a postnatal switch in functional properties of
neuromuscular transmission occurs when miniature end plate currents
become shorter and the conductance and Ca
2+
permeability of
end plate channels increases. These changes are due to replacement
during early neonatal development of the γ-subunit of the fetal
acetylcholine receptor (AChR) by the ɛ-subunit. The long-term
functional consequences of this switch for neuromuscular transmission
and motor behavior of the animal remained elusive. We report that
deletion of the ɛ-subunit gene caused in homozygous mutant mice the
persistence of γ-subunit gene expression in juvenile and adult
animals. Neuromuscular transmission in these animals is based on fetal
type AChRs present in the end plate at reduced density. Impaired
neuromuscular transmission, progressive muscle weakness, and atrophy
caused premature death 2 to 3 months after birth. The results
demonstrate that postnatal incorporation into the end plate of
ɛ-subunit containing AChRs is essential for normal development of
skeletal muscle.
A procedure for the prediction of temperature-sensitive mutants of a globular protein based solely on the amino acid sequence
Authors: Varadarajan R. , Nagarajaram H. A. , Ramakrishnan C.
Published: July 2002

Temperature-sensitive (Ts) mutants of a protein are an extremely
powerful tool for studying protein function
in vivo
and
in cell culture. We have devised a method to predict those residues in
a protein sequence that, when appropriately mutated, are most likely to
give rise to a Ts phenotype. Since substitutions of buried hydrophobic
residues often result in significant destabilization of the protein,
our method predicts those residues in the sequence that are likely to
be buried in the protein structure. We also indicate a set of amino
acid substitutions, which should be made to generate a Ts mutant of the
protein. This method requires only the protein sequence. No structural
information or homologous sequence information is required. This method
was applied to a test data set of 30 nonhomologous protein structures
from the Protein Data Bank. All of the residues predicted by the method
to be ≥95% buried were, in fact, buried in the protein crystal
structure. In contrast, only 50% of all hydrophobic residues in this
data set were ≥95% buried. This method successfully predicts several
known Ts and partially active mutants of T4 lysozyme, λ repressor,
gene V protein, and staphylococcal nuclease. This method also correctly
predicts residues that form part of the hydrophobic cores of λ
repressor, myoglobin, and cytochrome b562.
Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).
Authors: Liu C G , Maercker C , Castañon M J , Hauptmann R , Wiche G
Published: July 2002

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.
Capsule switching of
Neisseria meningitidis
Authors: Swartley J. S. , Marfin A. A. , Edupuganti S. , Liu L.-J. , Cieslak P. , Perkins B. , Wenger J. D. , Stephens D. S.
Published: July 2002

The different sialic acid (serogroups B, C, Y, and W-135) and nonsialic acid (serogroup A) capsular polysaccharides expressed by
Neisseria meningitidis
are major virulence factors and are used as epidemiologic markers and vaccine targets. However, the identification of meningococcal isolates with similar genetic markers but expressing different capsular polysaccharides suggests that meningococcal clones can switch the type of capsule they express. We identified, except for capsule, isogenic serogroups B [(α2→8)-linked polysialic acid] and C [(α2→9)-linked polysialic acid] meningococcal isolates from an outbreak of meningococcal disease in the U. S. Pacific Northwest. We used these isolates and prototype serogroup A, B, C, Y, and W-135 strains to define the capsular biosynthetic and transport operons of the major meningococcal serogroups and to show that switching from the B to C capsule in the outbreak strain was the result of allelic exchange of the polysialyltransferase. Capsule switching was probably the result of transformation and horizontal DNA exchange
in vivo
of a serogroup C capsule biosynthetic operon. These findings indicate that closely related virulent meningococcal clones may not be recognized by traditional serogroup-based surveillance and can escape vaccine-induced or natural protective immunity by capsule switching. Capsule switching may be an important virulence mechanism of meningococci and other encapsulated bacterial pathogens. As vaccine development progresses and broader immunization with capsular polysaccharide conjugate vaccines becomes a reality, the ability to switch capsular types may have important implications for the impact of these vaccines.
DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase
Authors: Kelleher Daniel J. , Gilmore Reid
Published: July 2002

DAD1, the
d
efender against
a
poptotic cell
d
eath, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithio
bis
(succinimidylpropionate). Crosslinked ribophorin II–OST48 heterodimers, DAD1–ribophorin II–OST48 heterotrimers and DAD1–ribophorin I–ribophorin II–OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.