bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of a dynamic
in vitro
attachment and invasion system (DIVAS) to determine influence of growth rate on invasion of respiratory epithelial cells by group B
Streptococcus
Authors: Malin Gennady , Paoletti Lawrence C.
Published: July 2002

Expression of capsular polysaccharide (CPS) and some surface proteins by group B
Streptococcus
(GBS) is regulated by growth rate. We hypothesized that precise control of GBS growth, and thus surface-expressed components, could modulate the ability of GBS to invade eukaryotic cells. To test this hypothesis, a dynamic
in vitro
attachment and invasion system (DIVAS) was developed that combines the advantages of bacterial growth in continuous culture with tissue culture. Tissue culture flasks were modified with inlet and outlet ports to permit perfusion of GBS. Encapsulated type III GBS strains M781 and COH1 and strains COH1-11 and COH1-13 (transposon mutants of COH1 that express an asialo CPS or are acapsular, respectively) were grown in continuous culture in a chemically defined medium at fast mass doubling time (
t

d

= 1.8 h) and slow (
t

d

= 11 h) growth rates, conditions previously shown to induce and repress, respectively, type III CPS expression. Encapsulated GBS strains invaded A549 respiratory epithelial cells 20- to 700-fold better at the fast than at the slow growth rate, suggesting a role for CPS. However, unencapsulated GBS were also invasive but only when cultured at the fast growth rate, which indicates that GBS invasion is independent of CPS expression and can be regulated by growth rate. Growth rate-dependent invasion occurred when GBS was grown in continuous culture under glucose-defined, thiamine-defined, and nondefined nutrient limitations. These results suggest a growth rate-dependent regulation of GBS pathogenesis and demonstrate the usefulness of DIVAS as a tool in studies of host–microbe interactions.
Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localization is regulated by oncogenic Ras
Authors: Zhou Xianbo , Richon Victoria M. , Wang Audrey H. , Yang Xiang-Jiao , Rifkind Richard A. , Marks Paul A.
Published: July 2002

Histone deacetylase 4 (HDAC4) is a member of a family of enzymes
that catalyze the removal of acetyl groups from core histones,
resulting in a compact chromatin structure that is generally associated
with repressed gene transcription. Protein phosphorylation has been
implicated in the regulation of the corepressor activity of the
deacetylase. Here we report that serine/threonine kinases are found
in association with HDAC4 and phosphorylate HDAC4
in
vitro
, and HDAC4 is phosphorylated in cells. The extracellular
signal-regulated kinases 1 and 2 (ERK1/2), also known as
p44
MAPK
and p42
MAPK
, respectively, are two of
the kinases associated with HDAC4. ERK1/2 are components of the
Ras–mitogen-activated protein kinase (MAPK) signal transduction
pathway. Activation of the Ras–MAPK pathway by expression of oncogenic
Ras or constitutively active MAPK/ERK kinase 1 results in an
increased percentage of cells (from ≈10% to ≈70%) that express
HDAC4 in the nucleus in C2C12 myoblast cells. In cells transfected with
oncogenic Ras, nuclear HDAC4 is associated with kinase activity. Our
results provide evidence that protein kinase activity is present in a
protein complex with HDAC4 and directly links the Ras–MAPK signal
transduction pathway to a mechanism for chromatin remodeling (i.e.,
histone deacetylation).
Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains
Authors: Malecki Marek , Hsu Annie , Truong Lynn , Sanchez Sylvia
Published: July 2002

To study the molecular structure and function of gene productsin situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 × 106M−1) and scFv:MBD (4.17 × 106M−1) were close to those of Fab proteolytic fragments (9.78 × 106M−1) derived from the parental IgG antibodies. After saturation of MBD with nickel or cobalt, scFv:MBD was imaged with electron spectroscopic imaging at each element's specific energy loss, thus generating the element's map. Immunolabeling with scFv:MBD resulted in a significant improvement of the labeling fidelity over that obtained with Fab or IgG derivatives, as it produced a much heavier specific labeling and label-free background. As determined with radioimmunoassay, labeling effectiveness with scFv:MBD was nearly the same as with scFv, but much higher than with scFv conjugated to colloidal gold, Nanogold, or horseradish peroxidase. This technology opens possibilities for simultaneous imaging of multiple molecules labeled with scFv:MBD at the molecular resolution within the same sample with electron spectroscopic imaging. Moreover, the same scFv:MBD can also be imaged with fluorescence resonance energy transfer and lifetime imaging as well as positron emission tomography and magnetic resonance imaging. Therefore, this technology may serve as an integrative factor in life science endeavors.
Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.
Authors: Murakami Y , Huberman J A , Hurwitz J
Published: July 2002

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.
Using ubiquitin to follow the metabolic fate of a protein.
Authors: Lévy F , Johnsson N , Rümenapf T , Varshavsky A
Published: July 2002

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.
Purification of the Drosophila RNA polymerase II general transcription factors.
Authors: Austin R J , Biggin M D
Published: July 2002

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.
prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors.
Authors: Nouwen N , de Kruijff B , Tommassen J
Published: July 2002

The SecY protein of Escherichia coli is an integral membrane component of the protein export apparatus. Suppressor mutations in the secY gene (prlA alleles) have been isolated that restore the secretion of precursor proteins with defective signal sequences. These mutations have never been shown to affect the translocation of wild-type precursor proteins. Here, we report that prlA suppressor mutations relieve the proton-motive force (pmf) dependency of the translocation of wild-type precursors, both in vivo and in vitro. Furthermore, the proton-motive force dependency of the translocation of a precursor with a stably folded domain in the mature region was suppressed by prlA mutations in vitro. These data show that prlA mutations cause a general relaxation of the export apparatus rather than a specific change that results in bypassing of the recognition of the signal sequence. In addition, these results are indicative for a mechanism in which the proton-motive force stimulates translocation by altering the conformation of the translocon.
Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.
Authors: Burfeind P , Chernicky C L , Rininsland F , Ilan J , Ilan J
Published: July 2002

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.
A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.
Authors: Yeakley J M , Morfin J P , Rosenfeld M G , Fu X D
Published: July 2002

A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.
Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11.
Authors: Liu W W , Mattingly R R , Garrison J C
Published: July 2002

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.
Glass transition in DNA from molecular dynamics simulations.
Authors: Norberg J , Nilsson L
Published: July 2002

Molecular dynamics simulations of the oligonucleotide duplex d(CGCGCG)2 in aqueous solution are used to investigate the glass transition phenomenon. The simulations were performed at temperatures in the 20 K to 340 K range. The mean square atomic fluctuations showed that the behavior of the oligonucleotide duplex was harmonic at low temperatures. A glass transition temperature at 223 K to 234 K was inferred for the oligonucleotide duplex, which is in agreement with experimental observations. The largest number of hydrogen bounds between the polar atoms of the oligonucleotide duplex and the water molecules was obtained at the glass transition temperature. With increasing temperature we observed a decrease in the average lifetime of the hydrogen bonds to water molecules.
Initiation sites of protein folding by NMR analysis.
Authors: Freund S M , Wong K B , Fersht A R
Published: July 2002

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.
Mutations in the C-terminal fragment of DnaK affecting peptide binding.
Authors: Burkholder W F , Zhao X , Zhu X , Hendrickson W A , Gragerov A , Gottesman M E
Published: July 2002

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate.
Haemophilus influenzae pili are composite structures assembled via the HifB chaperone.
Authors: St Geme J W , Pinkner J S , Krasan G P , Heuser J , Bullitt E , Smith A L , Hultgren S J
Published: July 2002

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.
Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain
Authors: Morinaga Naoko , Tsai Su-Chen , Moss Joel , Vaughan Martha
Published: July 2002

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor
(ARF)-bound GDP for GTP by a Golgi-associated guanine
nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E.
(1992)
Nature (London)
360, 352–354; Donaldson, J. G.,
Finazzi, D. & Klausner, R. D. (1992)
Nature (London)
360,
350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after
purification from bovine brain and rat spleen, it was no longer
BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996)
Proc. Natl. Acad. Sci. USA
93, 305–309]. We describe
here purification from bovine brain cytosol of a BFA-inhibited GEP.
After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and
precipitation at pH 5.8, GEP was eluted from Superose 6 as a large
molecular weight complex at the position of thyroglobulin (≈670 kDa).
After SDS/PAGE of samples from column fractions, silver-stained
protein bands of ≈190 and 200 kDa correlated with activity.
BFA-inhibited GEP activity of the 200-kDa protein was demonstrated
following electroelution from the gel and renaturation by dialysis.
Four tryptic peptides from the 200-kDa protein had amino acid sequences
that were 47% identical to sequences in Sec7 from
Saccharomyces
cerevisiae
(total of 51 amino acids), consistent with the view
that the BFA-sensitive 200-kDa protein may be a mammalian counterpart
of Sec7 that plays a similar role in cellular vesicular transport and
Sec7 may be a GEP for one or more yeast ARFs.
Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene
Authors: Tucker Kerry Lee , Talbot Dale , Lee Min Ae , Leonhardt Heinrich , Jaenisch Rudolf
Published: July 2002

Previous attempts to express functional DNA cytosine
methyltransferase (EC
2.1.1.37
) in cells transfected with the available
Dnmt
cDNAs have met with little or no success. We show
that the published
Dnmt
sequence encodes an amino
terminal-truncated protein that is tolerated only at very low levels
when stably expressed in embryonic stem cells. Normal expression levels
were, however, obtained with constructs containing a continuation of an
ORF with a coding capacity of up to 171 amino acids upstream of the
previously defined start site. The protein encoded by these constructs
comigrated in SDS/PAGE with the endogenous enzyme and
restored methylation activity in transfected cells. This was shown by
functional rescue of
Dnmt
mutant embryonic stem cells
that contain highly demethylated genomic DNA and fail to differentiate
normally. When transfected with the minigene construct, the genomic DNA
became remethylated and the cells regained the capacity to form
teratomas that displayed a wide variety of differentiated cell types.
Our results define an amino-terminal domain of the mammalian MTase that
is crucial for stable expression and function
in vivo
.
Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules
Authors: Pierres Anne , Benoliel Anne Marie , Bongrand Pierre , van der Merwe P. Anton
Published: July 2002

We studied single molecular interactions between surface-attached
rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found
on antigen-presenting cells. Spherical particles were coated with
decreasing densities of CD48–CD4 chimeric molecules then driven along
CD2-derivatized glass surfaces under a low hydrodynamic shear rate.
Particles exhibited multiple arrests of varying duration. By analyzing
the dependence of arrest frequency and duration on the surface density
of CD48 sites, it was concluded that (
i
) arrests were
generated by single molecular bonds and (
ii
) the initial
bond dissociation rate was about 7.8 s
−1
. The force
exerted on bonds was increased from about 11 to 22 pN; the detachment
rate exhibited a twofold increase. These results agree with and extend
studies on the CD2–CD48 interaction by surface plasmon resonance
technology, which yielded an affinity constant of ≈10
4
M
−1
and a dissociation rate of ≥6 s
−1
. It is
concluded that the flow chamber technology can be an useful complement
to atomic force microscopy for studying interactions between isolated
biomolecules, with a resolution of about 20 ms and sensitivity of a few
piconewtons. Further, this technology might be extended to actual
cells.
Transforming neural computations and representing time
Authors: Hopfield J. J.
Published: July 2002

Motifs of neural circuitry seem surprisingly conserved over
different areas of neocortex or of paleocortex, while performing quite
different sensory processing tasks. This apparent paradox may be
resolved by the fact that seemingly different problems in sensory
information processing are related by transformations (changes of
variables) that convert one problem into another. The same basic
algorithm that is appropriate to the recognition of a known odor
quality, independent of the strength of the odor, can be used to
recognize a vocalization (e.g., a spoken syllable), independent of
whether it is spoken quickly or slowly. To convert one problem into the
other, a new representation of time sequences is needed. The time that
has elapsed since a recent event must be represented in neural
activity. The electrophysiological hallmarks of cells that are involved
in generating such a representation of time are discussed. The
anatomical relationships between olfactory and auditory pathways
suggest relevant experiments. The neurophysiological mechanism for the
psychophysical logarithmic encoding of time duration would be of direct
use for interconverting olfactory and auditory processing problems.
Such reuse of old algorithms in new settings and representations is
related to the way that evolution develops new biochemistry.
A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.
Authors: Loh S N , Rohl C A , Kiefhaber T , Baldwin R L
Published: July 2002

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.
Social evolution in a new environment: the case of introduced fire ants.
Authors: Ross K G , Vargo E L , Keller L
Published: July 2002

The inadvertent introduction of the fire ant Solenopsis invicta to the United States from South America provides the opportunity to study recent social evolution by comparing social organization in native and introduced populations. We report that several important elements of social organization in multiple-queen nests differ consistently and dramatically between ants in Argentina and the United States. Colonies in Argentina contain relatively few queens and they are close relatives, whereas colonies in the United States contain high numbers of unrelated queens. A corollary of these differences is that workers in the native populations are significantly related to the new queens that they rear in contrast to the zero relatedness between workers and new queens in the introduced populations. The observed differences in queen number and relatedness signal a shift in the breeding biology of the introduced ants that is predicted on the basis of the high population densities in the new range. An additional difference in social organization that we observed, greater proportions of permanently unmated queens in introduced than in native populations, is predicted from the loss of alleles at the sex-determining locus and consequent skewing of operational sex ratios in the colonizing ants. Thus, significant recent social evolution in fire ants is consistent with theoretical expectations based on the altered ecology and population genetics of the introduced populations.
A cellular 65-kDa protein recognizes the negative regulatory element of human papillomavirus late mRNA
Authors: Dietrich-Goetz Walter , Kennedy Iain M. , Levins Beth , Stanley Margaret A. , Clements J. Barklie
Published: July 2002

Papillomavirus late gene expression is tightly linked to the
differentiation state of the host cell. Levels of late mRNAs are only
in part controlled by regulation of the late promoter, other
posttranscriptional mechanisms exist that reduce the amount of late
mRNA in undifferentiated cells. Previously we described a negative
regulatory element (NRE) located upstream of the human papillomavirus
type 16 late poly(A) site. We have delineated the NRE to a 79-nt region
in which a G+U-rich region was the major determinant of NRE activity.
UV-crosslinking assays identified a prominent nuclear protein of 65 kDa
as the only factor in close contact with the NRE, and a complex of at
least five proteins, including the 65-kDa protein, was enriched on
NRE–RNA. Binding of the 65-kDa protein was depleted by preincubation
with poly(U) Sepharose in high salt, a property characteristic of the
U2 small nuclear ribonucleoprotein auxiliary factor U2AF
65
and bacterially expressed U2AF
65
exhibited NRE binding. The
65-kDa protein bound to the G+U-rich NRE 3′ half which shows homology
to the B2P2 sequence a known U2AF
65
binding site in the
α-tropomyosin gene, and the G+U-rich element can be replaced by B2P2
in the binding assay. Treatment of cells with phorbol 12-myristate
13-acetate reduced binding of the 65-kDa protein, induced NRE binding
of a cytoplasmic protein, and relieved the NRE block on reporter gene
expression.
Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae andRhizobium-induced nodules may be conserved
Authors: van Rhijn P. , Fang Y. , Galili S. , Shaul O. , Atzmon N. , Wininger S. , Eshed Y. , Lum M. , Li Y. , To V. , Fujishige N. , Kapulnik Y. , Hirsch A. M.
Published: July 2002

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40andMsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogenRhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels oftrans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses—nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.
The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations
Authors: Wu Xin-Qi , Gu Wei , Meng XiuHua , Hecht Norman B.
Published: July 2002

The mouse RNA-binding protein, TB-RBP, suppresses translationin vitroand attaches mRNAs to microtubules by binding to conserved elements in the 3′ untranslated regions of specific mRNAs. We have now purified TB-RBP from testicular and brain cytoplasmic extracts and cloned its cDNA. We find that the mouse TB-RBP cDNAs contain an open reading frame of 228 amino acids with a leucine zipper domain within its C terminus, a transmembrane helix, and a group of putative phosphorylation sites. TB-RBP shows 99% identity to the human protein, translin, a recombination hotspot-binding protein associated with chromosomal translocations [Aoki, K., Suzuki, K., Sugano, T., Tasaka, T., Nakahara, K., Kuge, O., Omori, A. & Kasai, M. (1995)Nat. Genet.10, 167–174]. As shown for translin, TB-RBP also binds to single-stranded DNAs containing a broad range of consensus sequences, many of which are similar to the Y and H RNA-binding sequences. Recombinant TB-RBP was synthesized and an antiserum was prepared against the recombinant protein. The identity between translin and TB-RBP was confirmed by demonstrating that immunoprecipitation of TB-RBP from testicular extracts abolished formation of the RNA-TB–RBP complex. Based upon its DNA binding to target sequences in clustered breakpoint regions, we propose that TB-RBP may be involved in DNA recombination or DNA repair in male germ cells.
Ca
2+
-independent and Ca
2+
/GTP-binding protein-controlled exocytosis in a plant cell
Authors: Homann Ulrike , Tester Mark
Published: July 2002

Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca
2+
and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 μM) free Ca
2+
and nonhydrolyzable guanine nucleotides guanosine 5′-γ-thio]triphosphate (GTP[γS]) or guanosine 5′-[β-thio]diphosphate (GDP[βS]). With less than 2 nM cytoplasmic free Ca
2+
, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[γS] or GDP[βS]. In contrast, dialyzing protoplasts with 1 μM free Ca
2+
resulted in a large increase in membrane capacitance that was slightly reduced by GTP[γS] and strongly inhibited by GDP[βS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca
2+
-independent and whose regulation is currently not known and another that is stimulated by Ca
2+
and modulated by GTP-binding proteins. We suggest that Ca
2+
-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca
2+
-stimulated exocytosis may play a role in gibberellic acid-stimulated α-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.
Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein
Authors: Zhang Yi , Allison James P.
Published: July 2002

CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201to F201mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.
ActA is a dimer
Authors: Mourrain Philippe , Lasa Iñigo , Gautreau Alexis , Gouin Edith , Pugsley Anthony , Cossart Pascale
Published: July 2002

ActA, a surface protein of
Listeria monocytogenes
, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various
L. monocytogenes
strains expressing different ActA variants demonstrated that the region spanning amino acids 97–126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by
L. monocytogenes
, involving the ActA dimer, is presented.
The dynamins: Redundant or distinct functions for an expanding family of related GTPases?
Authors: Urrutia Raul , Henley John R. , Cook Tiffany , McNiven Mark A.
Published: July 2002

In the 7 years since dynamin was first isolated from bovine brain in search of novel microtubule-based motors, our understanding of this enzyme has expanded significantly. We now know that brain dynamin belongs to a family of large GTPases, which mediate vesicle trafficking. Furthermore, this enzymatic activity is markedly increased through association with microtubules, acidic phospholipids, and certain regulatory proteins that contain Src homology 3 (SH3) domains. From functional, genetic, and cellular manipulations, it is now generally accepted that dynamin participates in the endocytic uptake of receptors, associated ligands, and plasma membrane following an exocytic event. These observations have confirmed at least one function of dynamin that was predicted from seminal studies on a pleiotropic mutant,shibirets(shits) inDrosophila melanogaster.Of equal interest is the finding that there are multiple dynamin gene products, including two that are expressed in a tissue-specific manner, and they share marked homology with a larger family of distinct but related proteins. Therefore, it is attractive to speculate that the different dynamins may participate in related cellular functions, such as distinct endocytic processes and even secretion. In turn, dynamin could play an important role in cell growth, cell spreading, and neurite outgrowth. The purpose of this review is to enumerate on the expansive dynamin literature and to discuss the nomenclature, expression, and putative functions of this growing and interesting family of proteins.
Implications of a possible clustering of highest-energy cosmic rays
Authors: Sigl Günter , Schramm David N. , Lee Sangjin , Hill Christopher T.
Published: July 2002

Recently, a possible clustering of a subset of observed ultra-high energy cosmic rays above ≃40 EeV (4 × 10
19
eV) in pairs near the supergalactic plane was reported. We show that a confirmation of this effect would provide information on the origin and nature of these events and, in case of charged primaries, imply interesting constraints on the extragalactic magnetic field. Possible implications for the most common models of ultra-high energy cosmic ray production in the literature are discussed.
Genghis Khan (Gek) as a putative effector forDrosophilaCdc42 and regulator of actin polymerization
Authors: Luo Liqun , Lee Tzumin , Tsai Linus , Tang Gale , Jan Lily Y. , Jan Yuh Nung
Published: July 2002

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector forDrosophilaCdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study thein vivofunction ofgek, we generated mutations in theDrosophila geklocus. Egg chambers homozygous forgekmutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-typegektransgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.
DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity
Authors: Boyle Jefferey S. , Silva Anabel , Brady Jamie L. , Lew Andrew M.
Published: July 2002

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.
An
Arabidopsis
mutant that requires increased calcium for potassium nutrition and salt tolerance
Authors: Liu Jiping , Zhu Jian-Kang
Published: July 2002

Potassium (K
+
) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K
+
nutrition and salt tolerance are poorly understood. We report here the identification of an
Arabidopsis thaliana
mutant,
sos3
(
s
alt-
o
verly-
s
ensitive
3
), which is hypersensitive to Na
+
and Li
+
stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The
sos3
mutation also renders the plant unable to grow on low K
+
. Surprisingly, increased extracellular Ca
2+
suppresses the growth defect of
sos3
plants on low K
+
or 50 mM NaCl. In contrast, high concentrations of external Ca
2+
do not rescue the growth of the salt-hypersensitive
sos1
mutant on low K
+
or 50 mM NaCl. Under NaCl stress,
sos3
seedlings accumulated more Na
+
and less K
+
than the wild type. Increased external Ca
2+
improved K
+
/Na
+
selectivity of both
sos3
and wild-type plants. However, this Ca
2+
effect in
sos3
is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the
SOS3
locus is essential for K
+
nutrition, K
+
/Na
+
selectivity, and salt tolerance in higher plants.
Novel pyridinium surfactants for efficient, nontoxic
in vitro
gene delivery
Authors: van der Woude Irene , Wagenaar Anno , Meekel Arthur A. P. , ter Beest Martin B. A. , Ruiters Marcel H. J. , Engberts Jan B. F. N. , Hoekstra Dick
Published: July 2002

Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as β-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (≈50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex.