bluuebunny/crossref_metadata_2025 huggingface dataset:
Memory landscapes of single-enzyme molecules
Authors: Edman Lars , Rigler Rudolf
Published: July 2002

Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active.
Surgically created neural pathways mediate visual pattern discrimination
Authors: Frost Douglas O. , Boire Denis , Gingras Guy , Ptito Maurice
Published: July 2002

Combined lesions of retinal targets and ascending auditory pathways
can induce, in developing animals, permanent retinal projections to
auditory thalamic nuclei and to visual thalamic nuclei that normally
receive little direct retinal input. Neurons in the auditory cortex of
such animals have visual response properties that resemble those of
neurons in the primary visual cortex of normal animals. Therefore, we
investigated the behavioral function of the surgically induced
retino-thalamo-cortical pathways. We showed that both surgically
induced pathways can mediate visually guided behaviors whose normal
substrate, the pathway from the retina to the primary visual cortex via
the primary thalamic visual nucleus, is missing.
Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers
Authors: Ozaki Shoichiro , DeWald Daryll B. , Shope Joseph C. , Chen Jian , Prestwich Glenn D.
Published: July 2002

Phosphoinositide signaling regulates events in endocytosis and
exocytosis, vesicular trafficking of proteins, transduction of
extracellular signals, remodeling of the actin cytoskeleton, regulation
of calcium flux, and apoptosis. Obtaining mechanistic insights
in living cells is impeded by the membrane impermeability of these
anionic lipids. We describe a carrier system for intracellular delivery
of phosphoinositide polyphosphates (PIP
n
s) and
fluorescently labeled PIP
n
s into living cells, such that
intracellular localization can be directly observed. Preincubation of
PIP
n
s or inositol phosphates with carrier polyamines
produced complexes that entered mammalian, plant, yeast, bacterial, and
protozoal cells in seconds to minutes via a nonendocytic mechanism.
Time-dependent transit of both PIP
n
s and the carrier to
specific cytosolic and nuclear compartments was readily visualized by
fluorescence microscopy. Platelet-derived growth factor treatment of
NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol
4,5-bisphosphate
[PtdIns(4,5)P
2
]-7-nitrobenz-2-oxa-1,3-diazole resulted in
the redistribution of the fluorescent signal, suggesting that
fluorescent PtdIns(4,5)P
2
was a substrate for phospholipase
C. We also observed a calcium flux in NIH 3T3 cells when complexes of
carrier and PtdIns(4,5)P
2
or inositol 1,4,5-trisphosphate
were added extracellularly. This simple intracellular delivery system
allows for the efficient translocation of biologically active
PIP
n
s, inositol phosphates, and their fluorescent
derivatives into living cells in a physiologically relevant context.
A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation
Authors: Kossel Albrecht H. , Cambridge Sidney B. , Wagner Uta , Bonhoeffer Tobias
Published: July 2002

Neurotrophins have been shown to be involved in functional
strengthening of central nervous system synapses. Although their
general importance in this process is undisputed, it remains unresolved
whether neurotrophins are truly mediators of synaptic strengthening or
merely important cofactors. To address this question, we have devised a
method to inactivate endogenous brain-derived neurotrophic
factor (BDNF) with high time resolution by “caging” a
function-blocking mAb against BDNF with a photosensitive protecting
compound. Different assays were used to show that this inactivation of
the Ab is reversible by UV light. Synaptic potentiation after
τ-burst stimulation in the CA1 region of acute hippocampal slices
was significantly less when applying the unmodified Ab compared with
the caged Ab. Importantly, photoactivation of the caged Ab during
the time of induction of synaptic enhancement led to a marked decrease
in potentiation. Our experiments therefore strengthen the view that
endogenous BDNF has fast effects during induction of
synaptic plasticity. The results additionally show that caged Abs can
provide a tool for precise spatiotemporal control over
endogenous protein levels.
Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease
Authors: Abrams C. K. , Bennett M. V. L. , Verselis V. K. , Bargiello T. A.
Published: July 2002

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell–cell channels in paired
Xenopus
oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca
2+
, and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (
i
) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (
ii
) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca
2+
concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.
Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.
Authors: Unson C G , Cypess A M , Wu C R , Goldsmith P K , Merrifield R B , Sakmar T P
Published: July 2002

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.
Functional specificity of Hoxa-4 in vertebral patterning lies outside of the homeodomain.
Authors: Sreenath T L , Pollock R A , Bieberich C J
Published: July 2002

The Hox family of proteins plays a central role in establishing the body plan of a wide range of metazoan organisms. Each member of this family of transcriptional regulators has a distinct functional specificity, yet they bind to similar DNA target sequences through their conserved homeodomain. The mechanisms whereby Hox proteins achieve their diverse specificities in vivo remain undefined. Using the opposing effects of Hoxa-4 and Hoxc-8 in vertebral patterning, we demonstrate by replacing the homeodomain of Hoxa-4 with that of Hoxc-8 that the functional specificity of Hoxa-4 does not track with the homeodomain. These observations provide evidence that other regions of Hox proteins play an important role in mediating functional specificity during mammalian embryogenesis.
Phosducin is a ubiquitous G-protein regulator.
Authors: Danner S , Lohse M J
Published: July 2002

Phosducin is a 33-kDa cytosolic regulator of G-protein-mediated signaling that has previously been thought to be specific for retina and pineal gland. In this study, we show widespread tissue distribution of phosducin by the amplification of its cDNA and the detection of two different transcripts in Northern analyses in liver, lung, heart, brain, and retina. On the protein level, phosducin could be detected in 12 bovine tissues by immune precipitation and subsequent Western analysis using anti-phosducin antibodies generated in two different species. Masking of phosducin in direct Western blots appears to explain the failure to detect phosducin in earlier studies. The concentration of phosducin in bovine brain was calculated in the range of 10 pmol/mg total cytosolic protein (approximately 1 microM), whereas in the other tissues, it was slightly less. In these concentrations, phosducin inhibited receptor-stimulated adenylyl cyclase activity in cell membranes by about 50%. Taken together, our results indicate that phosducin is a ubiquitous regulator of G-protein function.
A fusion pore phenotype in mast cells of the ruby-eye mouse
Authors: Oberhauser Andres F. , Fernandez Julio M.
Published: July 2002

Using patch-clamp capacitance and amperometric techniques, we have
identified an exocytotic phenotype that affects the function of the
fusion pore, the molecular structure that connects the lumen of a
secretory vesicle with the extracellular environment during exocytosis.
Direct observation of individual exocytotic events in mast cells from
the ruby-eye mouse (
ru
/
ru
) showed a
3-fold increase in the fraction and duration of transient fusion events
with respect to wild-type mice. The fraction of the total fusion events
that were transient increased from 0.22 ± 0.02 (wild type) to
0.65 ± 0.02 (
ru
/
ru
), and the
average duration of these events increased from 418 ± 32 ms (wild
type) to 1207 ± 89 ms (
ru
/
ru
). We
also show that this phenotype can reduce and delay an evoked secretory
response by causing the fusion of vesicles that have been previously
emptied by repeated cycles of transient fusion. The exocytotic
phenotype that we describe here may be a cause of diseases like
platelet storage pool deficiency and prolonged bleeding times for which
the ruby-eye mouse serves as an animal model. Furthermore, the
identification of the gene causing the fusion pore phenotype reported
here will illuminate the molecular mechanisms regulating exocytotic
fusion.
Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions
Authors: Tribet Christophe , Audebert Roland , Popot Jean-Luc
Published: July 2002

Amphipols are a new class of surfactants that make it
possible to handle membrane proteins in detergent-free aqueous solution
as though they were soluble proteins. The strongly hydrophilic backbone
of these polymers is grafted with hydrophobic chains, making them
amphiphilic. Amphipols are able to stabilize in aqueous solution under
their native state four well-characterized integral membrane proteins:
(
i
) bacteriorhodopsin,
(
ii
) a bacterial photosynthetic reaction center,
(
iii
) cytochrome
b
6
f
, and
(
iv
) matrix porin.
Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.
Authors: Isozaki U , Mitch W E , England B K , Price S R
Published: July 2002

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.
Neural tube defects and abnormal brain development in F52-deficient mice.
Authors: Wu M , Chen D F , Sasaoka T , Tonegawa S
Published: July 2002

F52 is a myristoylated, alanine-rich substrate for protein kinase C. We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina bifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects. Exencephaly is the prominent type of defect and leads to high prenatal lethality. Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects. Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.
Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.
Authors: Rudolph U , Spicher K , Birnbaumer L
Published: July 2002

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.
BCL2 regulates neural differentiation.
Authors: Zhang K Z , Westberg J A , Hölttä E , Andersson L C
Published: July 2002

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.
The heat shock-induced hyperphosphorylation of τ is estrogen-independent and prevented by androgens: Implications for Alzheimer disease
Authors: Papasozomenos Sozos Ch.
Published: July 2002

We have shown that heat shock induces rapid dephosphorylation of τ in both female and male rats followed by hyperphosphorylation only in female rats. To investigate the role of gonadal hormones, rats were ovariectomized (OVX), orchiectomized (ORX), or sham-gonadectomized and received replacement therapy with estradiol benzoate (EB), testosterone propionate (TP), or sesame oil (SO) vehicle for 2–3 weeks, respectively. At 0, 3, 6, and 12 hr after heat shock, immunoblot analysis of SDS cerebral extracts was performed using phosphate-dependent and -independent anti-τ antibodies. Seven groups of rats were analyzed: (
i
) sham-OVX + SO; (
ii
) OVX + SO; (
iii
) OVX + EB; (
iv
) sham-ORX + SO; (
v
) ORX + SO; (
vi
) ORX + TP; and (
vii
) ORX. In all seven groups, there was dephosphorylation of τ at 0 hr after heat shock. In all three groups of female rats, there was hyperphosphorylation of τ at 3 hr after heat shock, and its degree and temporal pattern were identical between the OVX + SO and OVX + EB groups. In male rats, there was hyperphosphorylation of τ at 3 hr after heat shock in both ORX + SO and ORX groups, and its degree was reduced in the ORX + TP group. Thus, dephosphorylation of τ is gonadal hormone-independent, but while its hyperphosphorylation is estrogen-independent it is prevented by androgens. Because τ is abnormally hyperphosphorylated in Alzheimer disease, which is more frequent in women than men, these findings suggest that androgens may exert a neuroprotective effect.
Histone underacetylation is an ancient component of mammalian X chromosome inactivation
Authors: Wakefield Matthew J. , Keohane Ann M. , Turner Bryan M. , Graves Jennifer A. Marshall
Published: July 2002

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Here we demonstrate differential histone H4 acetylation between the X chromosomes of a female marsupial,
Macropus eugenii
. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals.
Transcriptional regulation of the
Xlim-1
gene by activin is mediated by an element in intron I
Authors: Rebbert Martha L. , Dawid Igor B.
Published: July 2002

The
Xlim-1
gene is activated in the late blastula
stage of
Xenopus
embryogenesis in the mesoderm, and its
RNA product becomes concentrated in the Spemann organizer at early
gastrula stage. A major regulator of early expression of
Xlim-1
is activin or an activin-like signal. We report
experiments aiming to identify the activin response element in the
Xlim-1
gene. The 5′ flanking region of the gene contains
a constitutive promoter that is not activin responsive, whereas
sequences in the first intron mediate repression of basal promoter
activity and stimulation by activin. An intron-derived fragment of 212
nt is the smallest element that could mediate activin responsiveness.
Nodal and act-Vg1, factors with signaling properties similar to
activin, also stimulated
Xlim-1
reporter constructs,
whereas BMP-4 did not stimulate or repress the constructs. The
mechanism of activin regulation of
Xlim-1
and the
sequence of the response element are distinct from activin response
elements of other genes studied so far.
Design, synthesis, and
in vitro
evaluation of cytotoxic analogs of bombesin-like peptides containing doxorubicin or its intensely potent derivative, 2-pyrrolinodoxorubicin
Authors: Nagy Attila , Armatis Patricia , Cai Ren-Zhi , Szepeshazi Karoly , Halmos Gabor , Schally Andrew V.
Published: July 2002

Five peptide fragments, based on the C-terminal sequence of bombesin (BN)-(6-14) or BN-(7-14), were selected as carriers for radicals doxorubicin (DOX) and 2-pyrrolino-DOX to create hybrid cytotoxic analogs. All these compounds had a reduced peptide bond (CH
2
-NH or CH
2
-N) between positions 13 (Phe or Leu) and 14 (Phe, Leu, or Tac) (Tac = thiazolidine-4-carboxylic acid). Three pseudononapeptide carriers contained N-terminal
d
-Phe or
d
-Tpi at position 6 (Tpi = 2,3,4,9-tetrahydro-1
H
-pyrido[3,4-
b
]indole-3-carboxylic acid). Two pseudooctapeptides had Gln
7
at the N terminus. The conjugation of
N
-(9-fluorenylmethoxycarbonyl)doxorubicin (
N
-Fmoc-DOX)-14-
O
-hemiglutarate to the peptide carriers at the N terminus resulted in cytotoxic hybrids of BN-like peptides containing DOX. These hybrids could then be converted to analogs with 2-pyrrolino-DOX by a reaction with 4-iodobutyraldehyde. The ability of the carriers and the conjugates to inhibit the binding of
125
I-labeled [Tyr
4
]BN to receptors for BN/gastrin releasing peptide (GRP) on Swiss 3T3 cells was determined. Cytotoxic conjugates of pseudooctapeptide carrier analogs displayed the highest binding affinity (
K
D
≈1 nM). The cytotoxic BN analogs and their corresponding cytotoxic radicals exerted similar inhibitory effects on the
in vitro
growth of CFPAC-1 human pancreatic cancer, DMS-53 human lung cancer, PC-3 human prostate cancer, and MKN-45 human gastric cancer cell lines that have receptors for BN/GRP. In DMS-53 cells, the activity of 2-pyrrolino-DOX and its conjugates was ≈2500 times higher than that of DOX and its hybrids. These highly potent cytotoxic analogs of BN have been designed as targeted anti-tumor agents for the treatment of various cancers that possess receptors for BN/GRP.
The signal recognition particle receptor of
Escherichia coli
(FtsY) has a nucleotide exchange factor built into the GTPase domain
Authors: Moser Claudio , Mol Olaf , Goody Roger S. , Sinning Irmgard
Published: July 2002

Targeting of many secretory and membrane proteins to the inner
membrane in
Escherichia coli
is achieved by the signal
recognition particle (SRP) and its receptor (FtsY). In
E.
coli
SRP consists of only one polypeptide (Ffh), and a 4.5S
RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain)
with an α-helical domain on its N terminus (N domain). The nucleotide
binding kinetics of the NG domain of the SRP receptor FtsY have been
investigated, using different fluorescence techniques. Methods to
describe the reaction kinetically are presented. The kinetics of
interaction of FtsY with guanine nucleotides are quantitatively
different from those of other GTPases. The intrinsic guanine nucleotide
dissociation rates of FtsY are about 10
5
times higher than
in Ras, but similar to those seen in GTPases in the presence of an
exchange factor. Therefore, the data presented here show that the NG
domain of FtsY resembles a GTPase–nucleotide exchange factor complex
not only in its structure but also kinetically. The I-box, an insertion
present in all SRP-type GTPases, is likely to act as an intrinsic
exchange factor. From this we conclude that the details of the GTPase
cycle of FtsY and presumably other SRP-type GTPases are fundamentally
different from those of other GTPases.
Phentolamine block of K
ATP
channels is mediated by Kir6.2
Authors: Proks Peter , Ashcroft Frances M.
Published: July 2002

The ATP-sensitive K
+
-channel (K
ATP
channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca
2+
entry and insulin release. The β cell K
ATP
channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K
+
-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit K
ATP
channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. K
ATP
channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac K
ATP
channel.
Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity
Authors: Wang Ze , Jiang He , Yang Zhi-Qiong , Chacko Samuel
Published: July 2002

It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an
in vitro
motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDΔ24–53 lacking the residues 24–53 to myosin compared with the level of binding of CaDΔ54–85 missing the adjacent residues 54–85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24–53 nearly abolished the ability of CaD to inhibit actin filament velocity in the
in vitro
motility experiments, whereas CaDΔ54–85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1–597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the
in vitro
motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.
Protein kinase C as a signal for exocytosis
Authors: Billiard Julia , Koh Duk-Su , Babcock Donner F. , Hille Bertil
Published: July 2002

We have studied signaling mechanisms that stimulate exocytosis and luteinizing hormone secretion in isolated male rat pituitary gonadotropes. As judged by reverse hemolytic plaque assays, phorbol-12-myristate-13-acetate (PMA) stimulates as many gonadotropes to secrete as does gonadotropin-releasing hormone (GnRH). However, PMA and GnRH use different signaling pathways. The secretagogue action of GnRH is not very sensitive to bisindolylmaleimide I, an inhibitor of protein kinase C, but is blocked by loading cells with a calcium chelator, 1,2-
bis
-(2-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid. The secretagogue action of PMA is blocked by bisindolylmaleimide I and is not very sensitive to the intracellular calcium chelator. GnRH induces intracellular calcium elevations, whereas PMA does not. As judged by amperometric measurements of quantal catecholamine secretion from dopamine- or serotonin-loaded gonadotropes, the secretagogue action of PMA develops more slowly (in several minutes) than that of GnRH. We conclude that exocytosis of secretory vesicles can be stimulated independently either by calcium elevations or by activation of protein kinase C.
On the spurious endoproteolytic processing of the presenilin proteins in cultured cells and tissues
Authors: Dewji Nazneen N. , Do Chau , Singer S. J.
Published: July 2002

It has been widely reported that the presenilin proteins PS-1 and PS-2 in extracts derived from a variety of cultured cells and from tissues are fragmented extensively by endoproteolytic processing events. It generally has been presumed that this endoproteolysis is a physiologically normal intracellular event following presenilin expression, which might play an important role in the still unknown functions of these molecules in connection with Alzheimer disease. We demonstrate herein, however, that, if a variety of cultured cells and several mouse tissues are examined under conditions minimizing cell trauma, the presenilin molecules in the extracts are found to be intact but that, if the cells and tissues are prepared under somewhat more stressful conditions, the endoproteolytic fragments are then observed. We conclude that these particular endoproteolytic events are not the result of physiologically normal processing of the presenilins but are rather artifacts occurring during the common procedures of specimen preparation.
A genetic system to identify DNA polymerase β mutator mutants
Authors: Washington Stacy L. , Yoon Margaret S. , Chagovetz Alexander M. , Li Shu-Xia , Clairmont Caroline A. , Preston Bradley D. , Eckert Kristin A. , Sweasy Joann B.
Published: July 2002

DNA polymerase β (pol β) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol β mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol β to substitute for
Escherichia coli
DNA polymerase I in DNA replication
in vivo
. Using this screen, we identified 13 candidate pol β mutator mutants. Three of the candidate mutator mutants were further characterized
in vivo
and shown to confer an increased spontaneous mutation frequency over that of wild-type pol β to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol β-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol β-14 and another of our mutant proteins, pol β-166, is probably critical for accurate DNA synthesis by pol β. Thus, our genetic method of screening for pol β mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol β enzyme.
Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding
Authors: Huang Hsien-Bin , Horiuchi Atsuko , Goldberg Jonathan , Greengard Paul , Nairn Angus C.
Published: July 2002

Site-directed mutagenesis of selected residues of mammalian protein phosphatase 1 (PP-1) has been carried out to further define the mechanism of catalysis, activation by divalent cations, and inhibition by toxins and inhibitory proteins. Mutation of active site residues predicted to bind metals (N124D and H248N) resulted in a large loss of enzyme activity and decreased affinity for metal ions; mutation of residues predicted to bind phosphosubstrate (R96A or R221S) led to a large loss of enzyme activity; and mutation of active site residues (D95A and D208A) resulted in a large loss of enzyme activity. Mutants N124D, H248N, R96A, and R221S exhibited large decreases in sensitivity to the toxins calyculin A, okadaic acid, and microcystin and to thiophospho-DARPP-32. Mutation of Y272 (Y272F) had little effect on activity but resulted in a large decrease in sensitivity to okadaic acid and calyculin A. Mutant D208A exhibited a decrease in sensitivity to okadaic acid and calyculin A, but, paradoxically, the sensitivity to inhibition by thiophospho-DARPP-32 was increased. Mutation of acidic groove residues (E256R, E275R, E252A:D253A, and E252A:D253A:E256R) exhibited little change in enzyme activity and no change in sensitivity to toxins, but increased sensitivity to thiophospho-DARPP-32. These results suggest that toxins and phospho-DARPP-32 interact at the active site of PP-1 in a similar fashion despite their differences in structure. In addition, acidic groove residues appear to influence the interaction of the phosphoinhibitor with the active site of PP-1.
Chlorophyll
a
availability affects
psbA
translation and D1 precursor processing
in vivo
in
Synechocystis
sp. PCC 6803
Authors: He Qingfang , Vermaas Wim
Published: July 2002

Transcript accumulation and translation of
psbA
as well as processing of the D1 precursor protein were investigated in relation to chlorophyll availability
in vivo
in cyanobacterial strains lacking photosystem I (PS I). The
psbA
transcript level was almost independent of chlorophyll availability and was ≈3-fold lower in darkness than in continuous light (5 μE m
−2
s
−1
). Upon illumination, it reached a steady–state level within several hours. Upon growth under light-activated heterotrophic growth conditions (LAHG) in the PS I-less strain, D1 synthesis occurred immediately upon illumination. However, in PS I-less/
chlL
−
cells, which lacked the light-independent chlorophyll biosynthesis pathway and had very low chlorophyll levels after LAHG growth, very little D1 synthesis occurred upon illumination, and the synthesis rate increased with time. This result suggests a translational control of D1 biosynthesis related to chlorophyll availability. Upon illumination, initially a high level of the nonprocessed D1 precursor was observed by pulse labeling and immunodetection in LAHG-grown PS I-less/

chlL
−

cells but not in PS I-less cells. A significant amount of the D1 precursor eventually was processed to mature D1, and the half-life of the D1 precursor decreased as the chlorophyll content of the cells increased. The D1 processing enzyme CtpA was found to be present at similar levels regardless illumination or chlorophyll levels. We conclude that, directly or indirectly, chlorophyll availability is needed for D1 translation as well as for efficient processing of the D1 precursor.
Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca
2+
and K
+
channels
Authors: Li-Smerin Yingying , Swartz Kenton J.
Published: July 2002

Protein toxins from venomous animals exhibit remarkably specific and selective interactions with a wide variety of ion channels. Hanatoxin and grammotoxin are two related protein toxins found in the venom of the Chilean Rose Tarantula,
Phrixotrichus spatulata
. Hanatoxin inhibits voltage-gated K
+
channels and grammotoxin inhibits voltage-gated Ca
2+
channels. Both toxins inhibit their respective channels by interfering with normal operation of the voltage-dependent gating mechanism. The sequence homology of hanatoxin and grammotoxin, as well as their similar mechanism of action, raises the possibility that they interact with the same region of voltage-gated Ca
2+
and K
+
channels. Here, we show that each toxin can interact with both voltage-gated Ca
2+
and K
+
channels and modify channel gating. Moreover, mutagenesis of voltage-gated K
+
channels suggests that hanatoxin and grammotoxin recognize the same structural motif. We propose that these toxins recognize a voltage-sensing domain or module present in voltage-gated ion channels and that this domain has a highly conserved three-dimensional structure.
Angiotensin II stimulates vesicular H
+
-ATPase in rat proximal tubular cells
Authors: Wagner Carsten A. , Giebisch Gerhard , Lang Florian , Geibel John P.
Published: July 2002

Two mechanisms of H
+
ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H
+
ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na
+
/H
+
exchanger and the basolateral Na
+
/HCO
3
−
cotransporter. In the present study, we examined the effects of AII on H
+
-ATPase activity in isolated proximal tubule fragments. H
+
-ATPase activity was assessed by monitoring intracellular pH after Na
+
removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A
1
, removal of Cl
−
, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na
+
and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A
1
, by removal of Cl
−
, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl
−
channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H
+
-ATPase by a Cl
−
-dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO
3
−
reabsorption along the proximal tubule when tubules are exposed to AII.
Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: Studies
in vitro
, in animals, and in humans
Authors: Macallan Derek C. , Fullerton Catherine A. , Neese Richard A. , Haddock Katherine , Park Sunny S. , Hellerstein Marc K.
Published: July 2002

A method for measuring DNA synthesis and, thus, cell proliferation,
in vivo
is presented. The technique consists of administering [6,6-
2
H
2
]Glc or [U-
13
C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [
3
H]thymidine or BrdUrd techniques in that the
de novo
nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here
in vitro
to the growth of HepG
2
and H
9
cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.
An essential role of phosphatidylinositol 3-kinase in myogenic differentiation
Authors: Jiang Bing-Hua , Zheng Jenny Z. , Vogt Peter K.
Published: July 2002

The oncogenep3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis.
Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin
Authors: Xiao Ming , Li Handong , Snyder Gregory E. , Cooke Roger , Yount Ralph G. , Selvin Paul R.
Published: July 2002

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin’s enzymology. The distance between these sites is 66.8 ± 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF
3
, (−1.6 ± 0.3 Å) and more significantly with ADP-AlF
4
(−4.6 ± 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP⋅P
i
is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP ≈ S1-ADP > S1-ADP-BeF
3
> S1-ADP-AlF
4
≈ S1-ADP-P
i
and that S1-ATP > S1-ADP-P
i
. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF
4
, and the post-powerstroke state, simulated by acto-S1-ADP.
Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells
Authors: Schwiebert Erik M. , Cid-Soto Luis P. , Stafford Diane , Carter Mark , Blaisdell Carol J. , Zeitlin Pamela L. , Guggino William B. , Cutting Garry R.
Published: July 2002

Cystic fibrosis (CF) is a lethal inherited disease that results from abnormal chloride conduction in epithelial tissues. ClC-2 chloride channels are expressed in epithelia affected by CF and may provide a key “alternative” target for pharmacotherapy of this disease. To explore this possibility, the expression level of ClC-2 channels was genetically manipulated in airway epithelial cells derived from a cystic fibrosis patient (IB3-1). Whole-cell patch-clamp analysis of cells overexpressing ClC-2 identified hyperpolarization-activated Cl
−
currents (HACCs) that displayed time- and voltage-dependent activation, and an inwardly rectifying steady-state current–voltage relationship. Reduction of extracellular pH to 5.0 caused significant increases in HACCs in overexpressing cells, and the appearance of robust currents in parental IB3-1 cells. IB3-1 cells stably transfected with the antisense ClC-2 cDNA showed reduced expression of ClC-2 compared with parental cells by Western blotting, and a significant reduction in the magnitude of pH-dependent HACCs. To determine whether changes in extracellular pH alone could initiate chloride transport via ClC-2 channels, we performed
36
Cl
−
efflux studies on overexpressing cells and cells with endogenous expression of ClC-2. Acidic extracellular pH increased
36
Cl
−
efflux rates in both cell types, although the ClC-2 overexpressing cells had significantly greater chloride conduction and a longer duration of efflux than the parental cells. Compounds that exploit the pH mechanism of activating endogenous ClC-2 channels may provide a pharmacologic option for increasing chloride conductance in the airways of CF patients.