bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic characterization of brush border myosin-I ATPase
Authors: Jontes James D. , Milligan Ronald A. , Pollard Thomas D. , Ostap E. Michael
Published: July 2002

Brush border myosin-I (BBM-I) is a single-headed unconventional
myosin found in the microvilli of intestinal epithelial cells. We used
stopped-flow kinetic analysis to measure the rate and equilibrium
constants for several steps in the BBM-I ATPase cycle. We determined
the rates for ATP binding to BBM-I and brush border actomyosin-I
(actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates
for the steps in ADP dissociation from actoBBM-I. The rate and
equilibrium constants for several of the steps in the actoBBM-I ATPase
are significantly different from those of other members of the myosin
superfamily. Most notably, dissociation of the actoBBM-I complex by ATP
and release of ADP from actoBBM-I are both very slow. The slow rates of
these steps may play a role in lengthening the time spent in
force-generating states and in limiting the maximal rate of BBM-I
motility. In addition, release of ADP from the actoBBM-I complex occurs
in at least two steps. This study provides evidence for a member of the
myosin superfamily with markedly divergent kinetic behavior.
Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers
Authors: Chanturiya Alexandr , Chernomordik Leonid V. , Zimmerberg Joshua
Published: July 2002

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological “fusion pores” can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.
An infectious arterivirus cDNA clone: Identification of a replicase point mutation that abolishes discontinuous mRNA transcription
Authors: van Dinten Leonie C. , den Boon Johan A. , Wassenaar Alfred L. M. , Spaan Willy J. M. , Snijder Eric J.
Published: July 2002

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated
in vitro
from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.
Analysis of highly disfavored processes through pathway-specific correlated fluorescence
Authors: Clair James J. La
Published: July 2002

A new method is described for the detection of disfavored reaction pathways. The approach combines organic synthesis, to independently prepare reactant and product, with the low detection limits of confocally adjusted fluorescence correlation spectroscopy. Selective detection of disfavored products is achieved by designing a system in which the analyte displays a unique absorption and emission of light. This was accomplished through application of a substance-selective intramolecular charge transfer. The power of this technique was demonstrated by monitoring the progress of the thermal retro-[2+2]-cycloaddition. In conjunction with concurrent
1
H-NMR monitoring, the relative abundance of major and disfavored reaction products can be determined and used to calculate the energetics of processes disfavored by more than 5 kcal/mol.
Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase
Authors: Townsley Fiona M. , Aristarkhov Alexander , Beck Sharon , Hershko Avram , Ruderman Joan V.
Published: July 2002

Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C
in vitro
. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the
in vitro
ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.
Jak1 kinase is required for cell migrations and anterior specification in zebrafish embryos
Authors: Conway Greg , Margoliath Ayelet , Wong-Madden Sharon , Roberts Richard J. , Gilbert Walter
Published: July 2002

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.
Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins
Authors: Chen Shuqi , Alon Ronen , Fuhlbrigge Robert C. , Springer Timothy A.
Published: July 2002

Antibodies immobilized on the wall of a flow chamber can support leukocyte rolling in shear flow. IgM mAb to Lewisx(CD15) and sialyl Lewisx(CD15s), which are carbohydrate antigens related to selectin ligands, plus mAb to CD48 and CD59, could mediate rolling. IgM and IgG mAb to L-selectin (CD62L), lymphocyte function-associated antigen 1 (CD11a), CD43, intercellular adhesion molecule 3 (CD50), and CD45 mediated only firm adhesion. In contrast to selectins, antibodies supported rolling only within a restricted range of site densities and wall shear stresses, outside of which firm adhesion or detachment occurred. When wall shear stress was increased, rolling velocity increased rapidly for antibodies but not for selectins. The kinetics of dissociation from the substrate of transiently tethered cells also increased more rapidly as a function of shear stress for antibodies than for selectins. These comparisons emphasize a number of remarkable features of selectins, including the lack of development of firm adhesion, and suggest that specialized molecular or cellular mechanisms must be required to explain their ability to support rolling over a wide range of environmental variables.
Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression
Authors: Kim Jin-Soo , Kim Jaesang , Cepek Karyn L. , Sharp Phillip A. , Pabo Carl O.
Published: July 2002

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h).
In vitro
transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.
Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity
Authors: Kawakami Yuko , Miura Toru , Bissonnette Reid , Hata Daisuke , Khan Wasif N. , Kitamura Toshio , Maeda-Yamamoto Mari , Hartman Stephen E. , Yao Libo , Alt Frederick W. , Kawakami Toshiaki
Published: July 2002

Mast cells derived from Bruton’s tyrosine kinase (Btk)-defectivexidorbtk nullmice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between thewtandxidmast cells,xidandbtk nullmast cells died by apoptosis more slowly than theirwtcounterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in thesebtk-mutated cells upon FcɛRI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected bybtkmutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution ofbtk nullmutant mast cells with thewt btkcDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK inwtmast cells, while JNK activity was consistently lower inbtk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.
Distinct forms of short-term plasticity at excitatory synapses of hippocampus and neocortex
Authors: Castro-Alamancos Manuel A. , Connors Barry W.
Published: July 2002

The expression of short- and long-term synaptic plasticity varies strongly across pathways in the central nervous system. Differences in the properties of transmitter release may underlie some of this variability. Here we compared the short-term plasticity displayed by a neocortical and a hippocampal pathway
in vitro
, and observed dramatic differences. Conditions known to increase transmitter release probability were more effective in hippocampus, while conditions known to decrease release probability were similarly effective in both pathways. The effects of the irreversible open-channel blocker of
N
-methyl-
d
-aspartate receptors, MK-801, implied that synapses in the neocortical pathway have a relatively high probability of transmitter release as compared with synapses in the hippocampal pathway. Differences in release probability may explain the pathway-specific variance in short- and long-term synaptic plasticity.
Fibroblast growth factor receptor 1 is fused to FIM in stem-cell myeloproliferative disorder with t(8;13)(p12;q12)
Authors: Popovici Cornel , Adélaïde José , Ollendorff Vincent , Chaffanet Max , Guasch Géraldine , Jacrot Michèle , Leroux Dominique , Birnbaum Daniel , Pébusque Marie-Josèphe
Published: July 2002

Chromosome 8p11–12 is the site of a recurrent breakpoint in a myeloproliferative disorder that involves lymphoid (T- or B-cell), myeloid hyperplasia and eosinophilia, and evolves toward acute leukemia. This multilineage involvement suggests the malignant transformation of a primitive hematopoietic stem cell. In this disorder, the 8p11–12 region is associated with three different partners 6q27, 9q33, and 13q12. We describe here the molecular characterization of the t(8;13) translocation that involves the
FGFR1
gene from 8p12, encoding a tyrosine kinase receptor for members of the fibroblast growth factor family, and a gene from 13q12, tentatively named
FIM
(
F
used
I
n
M
yeloproliferative disorders).
FIM
is related to
DXS6673E
, a candidate gene for X-linked mental retardation in Xq13.1; this defines a gene family involved in different human pathologies. The two reciprocal fusion transcripts,
FIM/FGFR1
and
FGFR1/FIM
are expressed in the malignant cells. The FIM/FGFR1 fusion protein contains the FIM putative zinc finger motifs and the catalytic domain of FGFR1. We show that it has a constitutive tyrosine kinase activity.
Parenting and survival in anthropoid primates: Caretakers live longer
Authors: Allman John , Rosin Aaron , Kumar Roshan , Hasenstaub Andrea
Published: July 2002

Most anthropoid primates are slow to develop, their offspring are mostly single births, and the interbirth intervals are long. To maintain a stable population, parents must live long enough to sustain the serial production of a sufficient number of young to replace themselves while allowing for the death of offspring before they can reproduce. However, in many species there is a large differential between the sexes in the care provided to offspring. Therefore, we hypothesize that in slowly developing species with single births, the sex that bears the greater burden in the care of offspring will tend to survive longer. Males are incapable of gestating infants and lactating, but in several species fathers carry their offspring for long periods. We predict that females tend to live longer than males in the species where the mother does most or all of the care of offspring, that there is no difference in survival between the sexes in species in which both parents participate about equally in infant care, and that in the species where the father does a greater amount of care than the mother, males tend to live longer. The hypothesis is supported by survival data for males and females in anthropoid primate species.
Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules
Authors: Zhang Lianshan , Torgerson Troy R. , Liu Xue-Yan , Timmons Sheila , Colosia Ann D. , Hawiger Jacek , Tam James P.
Published: July 2002

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH2-terminal 1,2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin β3, inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-κB, inhibited nuclear import of transcription factors NF-κB, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein–protein interactions.
The substrate-binding site in the lactose permease of
Escherichia coli
Authors: Venkatesan Pushpa , Kaback H. Ronald
Published: July 2002

Site-directed
N
-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 → Cys was also studied. In marked contrast to Val-331 → Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 → Ala/Val-331 → Cys, Arg-144 → Ala/Val-331 → Cys, or Arg-144 → Lys/Val-331 → Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 → Ala or Arg-144 → Ala and, to a lesser extent, Arg-144 → Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I
−
. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H
+
translocation pathway at the interface between helices IX and X.
A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons?
Authors: Ananiev E. V. , Phillips R. L. , Rines H. W.
Published: July 2002

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of
Zea mays
L. Comparative fluorescence
in situ
hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in
Drosophila
, maize knob DNA may have some properties of transposable elements.
Vesiculation and sorting from PC12-derived endosomes
in
vitro
Authors: Lichtenstein Yael , Desnos Claire , Faúndez Victor , Kelly Regis B. , Clift-O’Grady Lois
Published: July 2002

Formation of small vesicles resembling synaptic vesicles can be reconstituted
in vitro
by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998)
Cell
93, 423–432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15°C, but not at 4°C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor
in vitro
excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously
in vitro
with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.
Counting individual sulfur atoms in a protein by ultrahighresolution Fourier transform ion cyclotron resonance mass spectrometry: Experimental resolution of isotopic fine structure in proteins
Authors: Shi Stone D.-H. , Hendrickson Christopher L. , Marshall Alan G.
Published: July 2002

A typical molecular ion mass spectrum consists of a sum of signals from species of various possible isotopic compositions. Only the monoisotopic peak (e.g., all carbons are
12
C; all nitrogens are
14
N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of ∼1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two
13
C vs. two
15
N vs. one
13
C and one
15
N vs.
34
S, vs.
18
O, etc., at ∼2 Da higher in mass than the monoisotopic mass). At sufficiently high mass resolving power, each of these nominal-mass peaks resolves into its isotopic fine structure. Here, we report resolution of the isotopic fine structure of proteins up to 15.8 kDa (isotopic
13
C,
15
N doubly depleted tumor suppressor protein, p16), made possible by electrospray ionization followed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass analysis at 9.4 tesla. Further, a resolving power of m/Δm
50%
≈8,000,000 has been achieved on bovine ubiquitin (8.6 kDa). These results represent a 10-fold increase in the highest mass at which isotopic fine structure previously had been observed. Finally, because isotopic fine structure reveals elemental composition directly, it can be used to confirm or determine molecular formula. For p16, for example, we were able to determine (5.1 ± 0.3) the correct number (five) of sulfur atoms solely from the abundance ratio of the resolved
34
S peak to the monoisotopic peak.
Real time detection of DNA⋅RNA hybridization in living cells
Authors: Sokol Deborah L. , Zhang Xiaolin , Lu Ponzy , Gewirtz Alan M.
Published: July 2002

Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized “molecular beacon” (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5′ and 3′ ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem–loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem–loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be ≥1 × 10
−1
ag of MB, or ∼10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the
vav
protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that real-time visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.
Structural analysis of the nurse shark (new) antigen receptor (NAR): Molecular convergence of NAR and unusual mammalian immunoglobulins
Authors: Roux Kenneth H. , Greenberg Andrew S. , Greene Lesley , Strelets Lioudmila , Avila David , McKinney E. Churchill , Flajnik Martin F.
Published: July 2002

We recently have identified an antigen receptor in sharks called NAR (
n
ew or
n
urse shark
a
ntigen
r
eceptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (V
H
) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.
K
ATP
channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit
Authors: Drain Peter , Li Lehong , Wang Jing
Published: July 2002

ATP-sensitive potassium (“K
ATP
”) channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The K
ATP
channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (K
ir
6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic β cell K
ATP
channels expressed in
Xenopus
oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the K
ir
6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.
Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor)
Authors: Zhang Haidi , Serrero Ginette
Published: July 2002

The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the
in vivo
growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.
Testatin
: A cystatin-related gene expressed during early testis development
Authors: Töhönen Virpi , Österlund Christina , Nordqvist Katarina
Published: July 2002

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene
Sry
, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named
testatin
. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that
testatin
expression is restricted to fetal gonads and adult testis. Furthermore,
testatin
is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of
Sry
action, at a time immediately after the peak of
Sry
expression. This finding suggests that
testatin
might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.
Rapping, a female receptive call, initiates male–female duets in the South African clawed frog
Authors: Tobias Martha L. , Viswanathan Sandya S. , Kelley Darcy B.
Published: July 2002

Finding a sexually receptive partner of the opposite sex is a challenge; one solution is to advertise. That advertising is usually the province of males has shaped scenarios for sexual selection, especially the ardent active male courting the passive but choosy female. Herein we consider an unusual case in which constraints on reproduction may have led to fertility advertisement by female frogs. When oviposition is imminent, female South African clawed frogs swim to an advertising male and produce an aphrodisiac call, rapping, that stimulates both male vocalization and approach. Males respond to rapping with a distinctive answer call. The rapping–answer interaction thus forms a duet between partners of a receptive pair.
Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca
2+
channels expressed in dysgenic myotubes
Authors: Grabner Manfred , Dirksen Robert T. , Beam Kurt G.
Published: July 2002

Expression of cardiac L-type Ca
2+
channels in dysgenic myotubes results in large Ca
2+
currents and electrically evoked contractions resulting from Ca
2+
-entry dependent release of Ca
2+
from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca
2+
channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca
2+
currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca
2+
channels in close apposition to sarcoplasmic reticulum Ca
2+
release channels. We tagged the N termini of different α
1
subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged α
1
subunit exhibited Ca
2+
channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-α
1S
and GFP-α
1C
in dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-α
1A
and GFP-α
1B
failed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-α
1S
and GFP-α
1C
were present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-α
1A
and GFP-α
1B
were not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.
Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase
Authors: Rogatsky Inez , Logan Susan K. , Garabedian Michael J.
Published: July 2002

The mitogen-activated protein kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 phosphorylate and activate transcription factors that promote proliferative and inflammatory responses, whereas glucocorticoid receptor (GR) activation inhibits cell growth and inflammation. We demonstrate that JNK and ERK but not p38 phosphorylate GR
in vitro
primarily at Ser-246. Selective activation of either ERK or JNK
in vivo
inhibits GR-mediated transcriptional activation, which depends on receptor phosphorylation at Ser-246 by JNK but not ERK. Thus, JNK inhibits GR transcriptional activation by direct receptor phosphorylation, whereas ERK does so indirectly. We propose that phosphorylation of GR by JNK or of a GR cofactor by ERK provides mechanisms to ensure the rapid inhibition of GR-dependent gene expression when it conflicts with mitogenic or proinflammatory signals.
Competition among body parts in the development and evolution of insect morphology
Authors: Nijhout H. F. , Emlen D. J.
Published: July 2002

Changes in form during ontogeny and evolution depend in large measure on changes in the relative growth of the various parts of the body. The current consensus in developmental biology is that the final size of appendages and internal organs is regulated autonomously, within the structure itself. Size regulation of body parts typically requires no external control and is thought to be relatively insensitive to signals from the developmental environment. We show in two very different systems, butterfly wings and beetle horns, that experimentally induced changes in the allocation of developmental resources to one trait produces compensatory changes in the relative sizes of other traits. These findings illustrate that interaction among body parts in development is part of the mechanism of size regulation of those parts. Furthermore, in the case of beetle horns, we show that the tradeoff in size is manifest as a significant negative genetic correlation among the involved body parts and, therefore, constitutes a developmental source of genetic constraint on the evolution of body form.
R-type Ca
2+
currents evoke transmitter release at a rat central synapse
Authors: Wu Ling-Gang , Borst J. Gerard G. , Sakmann Bert
Published: July 2002

Voltage-dependent Ca
2+
currents evoke synaptic transmitter release. Of six types of Ca
2+
channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca
2+
channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca
2+
influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca
2+
channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca
2+
influx during a presynaptic action potential. This Ca
2+
current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca
2+
currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca
2+
channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.
Microtubule dysfunction by posttranslational nitrotyrosination of α-tubulin: A nitric oxide-dependent mechanism of cellular injury
Authors: Eiserich Jason P. , Estévez Alvaro G. , Bamberg Thaddeus V. , Ye Yao Zu , Chumley Phillip H. , Beckman Joseph S. , Freeman Bruce A.
Published: July 2002

NO
2
Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO
2
Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO
2
Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of α-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin–tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of α-tubulin, incorporation of NO
2
Tyr shows apparent irreversibility. Nitrotyrosination of α-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of α-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of α-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO
2
Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the α-tubulin tyrosination/detyrosination cycle plays in microtubule function.
Multiplex detection of four pathogenic retroviruses using molecular beacons
Authors: Vet Jacqueline A. M. , Majithia Amit R. , Marras Salvatore A. E. , Tyagi Sanjay , Dube Syamalima , Poiesz Bernard J. , Kramer Fred Russell
Published: July 2002

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100,000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.
Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions
Authors: Siess Wolfgang , Zangl Konrad J. , Essler Markus , Bauer Markus , Brandl Richard , Corrinth Carolin , Bittman Robert , Tigyi Gabor , Aepfelbacher Martin
Published: July 2002

Oxidized low density lipoprotein (LDL) is a key factor in the pathogenesis of atherosclerosis and its thrombotic complications, such as stroke and myocardial infarction. It activates endothelial cells and platelets through mechanisms that are largely unknown. Here, we show that lysophosphatidic acid (LPA) was formed during mild oxidation of LDL and was the active compound in mildly oxidized LDL and minimally modified LDL, initiating platelet activation and stimulating endothelial cell stress-fiber and gap formation. Antagonists of the LPA receptor prevented platelet and endothelial cell activation by mildly oxidized LDL. We also found that LPA accumulated in and was the primary platelet-activating lipid of atherosclerotic plaques. Notably, the amount of LPA within the human carotid atherosclerotic lesion was highest in the lipid-rich core, the region most thrombogenic and most prone to rupture. Given the potent biological activity of LPA on platelets and on cells of the vessel wall, our study identifies LPA as an atherothrombogenic molecule and suggests a possible strategy to prevent and treat atherosclerosis and cardiocerebrovascular diseases.
Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?
Authors: Bennett Gary J.
Published: July 2002

Painful peripheral neuropathies are precipitated by nerve injury
from disease or trauma. All such injuries will be accompanied by an
inflammatory reaction, a neuritis, that will mobilize the immune
system. The role of the inflammation itself is difficult to determine
in the presence of structural damage to the nerve. A method has been
devised to produce a focal neuritis in the rat sciatic nerve that
involves no more than trivial structural damage to the nerve. This
experimental focal neuritis produces neuropathic pain sensations (heat-
and mechano-hyperalgesia, and cold- and mechano-allodynia) in the
ipsilateral hind paw. The abnormal pain sensations begin in 1–2 days
and last for 4–6 days, with a subsequent return to normal. These
results suggest that there is a neuroimmune interaction that occurs at
the outset of nerve injury (and perhaps episodically over time in slow
developing conditions like diabetic neuropathy) that produces
neuropathic pain. The short duration of the phenomena suggest that they
may prime the system for more slowly developing mechanisms of abnormal
pain (e.g., ectopic discharge in axotomized primary afferent neurons)
that underlie the chronic phase of painful neuropathy.
Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments
Authors: Chakshusmathi G. , Ratnaparkhi Girish S. , Madhu P. K. , Varadarajan R.
Published: July 2002

Ordered protein complexes are often formed from partially ordered fragments that are difficult to structurally characterize by conventional NMR and crystallographic techniques. We show that concentration-dependent hydrogen exchange studies of a fragment complex can provide structural information about the solution structures of the isolated fragments. This general methodology can be applied to any bimolecular or multimeric system. The experimental system used here consists of Ribonuclease S, a complex of two fragments of Ribonuclease A. Ribonuclease S and Ribonuclease A have identical three-dimensional structures but exhibit significant differences in their dynamics and stability. We show that the apparent large dynamic differences between Ribonuclease A and Ribonuclease S are caused by small amounts of free fragments in equilibrium with the folded complex, and that amide exchange rates in Ribonuclease S can be used to determine corresponding rates in the isolated fragments. The studies suggest that folded RNase A and the RNase S complex exhibit very similar dynamic behavior. Thus cleavage of a protein chain at a single site need not be accompanied by a large increase in flexibility of the complex relative to that of the uncleaved protein.