bluuebunny/crossref_metadata_2025 huggingface dataset:
Aluminum activates an anion channel in the apical cells of wheat roots
Authors: Ryan Peter R. , Skerrett Martha , Findlay Geoffrey P. , Delhaize Emmanuel , Tyerman Stephen D.
Published: July 2002

We describe an anion channel in the plasmalemma of protoplasts isolated from wheat (
Triticum aestivum
L.) roots that is activated by aluminum (Al
3+
). In the whole-cell configuration, addition of 20–50 μM AlCl
3
to the external solution depolarized the membrane and activated an inward current that could remain active for more than 60 min. The activation by Al
3+
was rapid in 20% of protoplasts examined, whereas in another 30% a delay of more than 10 min occurred after Al
3+
was added. Once the current was activated, changing the external Cl
−
concentration shifted the membrane reversal potential with
E
Cl
, showing that the channel is more selective for anions than cations (Ca
2+
, K
+
, tetraethylammonium
+
). The channel could be activated by Al
3+
, but not by La
3+
, and was observed in protoplasts isolated from the root apex but not in protoplasts isolated from mature root tissue. The anion channel antagonist niflumate inhibited the current in whole cell measurements by 83% at 100 μM. Outside-out patch recordings revealed a multistate channel with single-channel conductances of between 27 and 66 pS.
The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5′-GCTGGTGG-3′
Authors: Bianco Piero R. , Kowalczykowski Stephen C.
Published: July 2002

The RecBCD enzyme ofEscherichia colifunctions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5′-GCTGGTGG-3′-containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.
In vivo
zippering of inner and outer mitochondrial membranes by a stable translocation intermediate
Authors: Schülke Norbert , Sepuri Naresh Babu V. , Pain Debkumar
Published: July 2002

It was previously assumed that the import of cytoplasmically synthesized precursor proteins into mitochondria occurs through a single structure spanning both outer and inner membranes at contact sites. Based on recent findings, however, the two membranes appear to contain independent translocation elements that reversibly cooperate during protein import. This feature makes it difficult to generate a means of isolating a fully integrated and functional translocation complex. To study these independent translocases
in vitro
and
in vivo
, we have constructed a chimeric protein consisting of an N-terminal authentic mitochondrial precursor (delta
1
-pyrroline-5-carboxylate dehydrogenase) linked, through glutathione
S
-transferase, to IgG binding domains derived from staphylococcal protein A. This construct becomes trapped
en route
to the matrix, spanning both outer and inner membranes in such a way that the entire signal-less delta
1
-pyrroline-5-carboxylate dehydrogenase moiety reaches the matrix, while only the folded protein A domain remains outside. During
in vivo
import of this precursor, outer and inner membranes of yeast mitochondria become progressively “zippered” together, forming long stretches of close contact. Using this novel intermediate, the outer and inner mitochondrial membrane channels, which normally interact only transiently, can be tightly joined (both
in vitro
and
in vivo
), forming a stable association. This suggests a method for isolating the functional translocation complex as a single entity.
Possible forcing of global temperature by the oceanic tides
Authors: Keeling Charles D. , Whorf Timothy P.
Published: July 2002

An approximately decadal periodicity in surface air temperature is
discernable in global observations from A.D. 1855 to 1900 and since
A.D. 1945, but with a periodicity of only about 6 years during the
intervening period. Changes in solar irradiance related to the sunspot
cycle have been proposed to account for the former, but cannot account
for the latter. To explain both by a single mechanism, we propose that
extreme oceanic tides may produce changes in sea surface temperature at
repeat periods, which alternate between approximately one-third and
one-half of the lunar nodal cycle of 18.6 years. These alternations,
recurring at nearly 90-year intervals, reflect varying slight degrees
of misalignment and departures from the closest approach of the Earth
with the Moon and Sun at times of extreme tide raising forces. Strong
forcing, consistent with observed temperature periodicities, occurred
at 9-year intervals close to perihelion (solar perigee) for several
decades centered on A.D. 1881 and 1974, but at 6-year intervals for
several decades centered on A.D. 1923. As a physical explanation for
tidal forcing of temperature we propose that the dissipation of extreme
tides increases vertical mixing of sea water, thereby causing episodic
cooling near the sea surface. If this mechanism correctly explains
near-decadal temperature periodicities, it may also apply to
variability in temperature and climate on other times-scales, even
millennial and longer.
Importin/karyopherin protein family members required for mRNA export from the nucleus
Authors: Seedorf Matthias , Silver Pamela A.
Published: July 2002

The yeast
Saccharomyces cerevisiae
contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa β subunit of the nuclear transport factor importin (also termed karyopherin and encoded by
KAP95/RSL1
in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin β (Kap95p/Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.
Loss of heterozygosity induced by a chromosomal double-strand break
Authors: Moynahan Mary Ellen , Jasin Maria
Published: July 2002

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (
Rb
) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the
Rb
locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.
Dominant transformation by mutated human
ras
genes
in vitro
requires more than 100 times higher expression than is observed in cancers
Authors: Hua Vivian Y. , Wang Wai K. , Duesberg Peter H.
Published: July 2002

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-
ras
, “play a dominant part in cancer,” because they are sufficient to transform transfected mouse cell lines
in vitro
[Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994)
Molecular Biology of the Cell
(Garland, New York)]. However, in cells transformed
in vitro
mutated human
ras
genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of
ras
transcription in cancers and the very high levels in cells transformed
in vitro
, we have investigated the minimal level of human
ras
expression for transformation
in vitro
. Using point-mutated human
ras
genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant
in vitro
transformation of the mouse C3H cell line only with
ras
genes linked to viral promoters. These
ras
genes were expressed more than 120-fold higher than are native
ras
genes of C3H cells. The copy number of transfected
ras
genes ranged from 2–6 in our system. In addition, nondominant transformation was observed in a small percentage (2–7%) of C3H cells transfected with
ras
genes that are expressed less than 20 times higher than native C3H
ras
genes. Because over 90% of cells expressing
ras
at this moderately enhanced level were untransformed, transformation must follow either a nondominant
ras
mechanism or a non-
ras
mechanism. We conclude that the mutated, but normally expressed,
ras
genes found in human and animal cancers are not likely to “play a dominant part in cancer.” The conclusion that mutated
ras
genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated
ras
in normal animals, and in benign human tissue, “which has little potential to progress” [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994)
Cancer Res.
54, 5523–5526]. Even the view that mutated
ras
is necessary for cancer is hard to reconcile with (
i
) otherwise indistinguishable cancers with and without
ras
mutations, (
ii
) metastases of the same human cancers with and without
ras
mutations, (
iii
) retroviral
ras
genes that are oncogenic without point mutations, and (
iv
) human tumor cells having spontaneously lost
ras
mutation but not tumorigencity.
RNA folding kinetics regulates translation of phage MS2 maturation gene
Authors: Poot Raymond A. , Tsareva Nina V. , Boni Irina V. , van Duin Jan
Published: July 2002

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model.
In vitro
experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.
The
ARS309
chromosomal replicator of
Saccharomyces cerevisiae
depends on an exceptional ARS consensus sequence
Authors: Theis James F. , Newlon Carol S.
Published: July 2002

Autonomously replicating sequence (ARS) elements, which
function as the cis-acting chromosomal replicators in the yeast
Saccharomyces cerevisiae
, depend upon an essential copy
of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the
chromosome III replicator
ARS309
unexpectedly revealed
that its essential ACS differs from the canonical ACS at two positions.
One of the changes observed in
ARS309
inactivates other
ARS elements. This atypical ACS binds the origin recognition complex
efficiently and is required for chromosomal replication origin
activity. Comparison of the essential ACS of
ARS309
with
the essential regions of other ARS elements revealed an expanded 17-bp
conserved sequence that efficiently predicts the essential core of ARS
elements.
Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness
Authors: Shah Nirao M. , Anderson David J.
Published: July 2002

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner,
in vitro
. Because NCSCs are likely exposed to multiple signals
in vivo
, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons,
in vivo
.
Role of lipid polymorphism in G protein-membrane interactions: Nonlamellar-prone phospholipids and peripheral protein binding to membranes
Authors: Escribá Pablo V. , Ozaita Andrés , Ribas Catalina , Miralles Antonio , Fodor Elfrieda , Farkas Tibor , García-Sevilla Jesús A.
Published: July 2002

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein α-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein β-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.
Construction of a Z-DNA-specific restriction endonuclease
Authors: Kim Yang-Gyun , Kim Pearl S. , Herbert Alan , Rich Alexander
Published: July 2002

Novel restriction enzymes can be created by fusing the nuclease domain ofFokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Zα) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Zα nuclease, which is a chimera of Zα andFokI nuclease. Purified Zα nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Zα nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Zα binds Z-DNA in an environment similar to that in a cell. Zα nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.
Regulation of number and size of digits by posterior
Hox
genes: A dose-dependent mechanism with potential evolutionary implications
Authors: Zákány József , Fromental-Ramain Catherine , Warot Xavier , Duboule Denis
Published: July 2002

The proper development of digits, in tetrapods, requires the activity of several genes of the
HoxA
and
HoxD
homeobox gene complexes. By using a variety of loss-of-function alleles involving the five
Hox
genes that have been described to affect digit patterning, we report here that the group 11, 12, and 13 genes control both the size and number of murine digits in a dose-dependent fashion, rather than through a
Hox
code involving differential qualitative functions. A similar dose–response is observed in the morphogenesis of the penian bone, the baculum, which further suggests that digits and external genitalia share this genetic control mechanism. A progressive reduction in the dose of
Hox
gene products led first to ectrodactyly, then to olygodactyly and adactyly. Interestingly, this transition between the pentadactyl to the adactyl formula went through a step of polydactyly. We propose that in the distal appendage of polydactylous short-digited ancestral tetrapods, such as Acanthostega, the
HoxA
complex was predominantly active. Subsequent recruitment of the
HoxD
complex contributed to both reductions in digit number and increase in digit length. Thus, transition through a polydactylous limb before reaching and stabilizing the pentadactyl pattern may have relied, at least in part, on asynchronous and independent changes in the regulation of
HoxA
and
HoxD
gene complexes.
A genetic screen for mutations that disrupt an auditory response in
Drosophila melanogaster
Authors: Eberl Daniel F. , Duyk Geoffrey M. , Perrimon Norbert
Published: July 2002

Hearing is one of the last sensory modalities to be subjected to genetic analysis in
Drosophila melanogaster
. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant,
5L3
, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail.
5L3
had a general courtship defect; courtship of females by
5L3
males also was affected strongly.
5P1
males courted females normally but had reduced success at copulation.
5P1
and
5N18
showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants,
5M8
and
5N30
, produced amotile sperm although in
5N30
this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants,
5G10
and
5N18
. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.
Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide
Authors: Zakhary Randa , Poss Kenneth D. , Jaffrey Samie R. , Ferris Christopher D. , Tonegawa Susumu , Snyder Solomon H.
Published: July 2002

Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3′,5′ monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOS
Δ
/
Δ
and HO-2
Δ
/
Δ
mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOS
Δ
/
Δ
animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2
Δ
/
Δ
animals.
Adult fast myosin pattern and Ca
2+
-induced slow myosin pattern in primary skeletal muscle culture
Authors: Kubis Hans-Peter , Haller Ernst-August , Wetzel Petra , Gros Gerolf
Published: July 2002

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca
2+
ionophore is added to the culture medium on day 11, raising intracellular [Ca
2+
] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs.
G
sα
-selective G protein antagonists
Authors: Hohenegger M. , Waldhoer M. , Beindl W. , Böing B. , Kreimeyer A. , Nickel P. , Nanoff C. , Freissmuth M.
Published: July 2002

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G
α
subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G
sα
. Two compounds have been identified: NF449 (4,4′,4",4′"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4,4′-[carbonylbis[imino-3,1-phenylene-(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (
i
) suppress the association rate of guanosine 5′-[γ-thio]triphosphate ([
35
S]GTP[γS]) binding to G
sα-s
but not to G
iα-1
, (
ii
) inhibit stimulation of adenylyl cyclase activity in S49 cyc
−
membranes (deficient in endogenous G
sα
) by exogenously added G
sα-s
, and (
iii
) block the coupling of β-adrenergic receptors to G
s
with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A
1
-adenosine receptor with its cognate G proteins (G
i
/G
o
) at concentrations that are >30-fold higher than those required for uncoupling of β-adrenergic receptor/G
s
tandems; similarly, the angiotensin II type-1 receptor (a prototypical G
q
-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for G
sα
-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.
Stepwisein vitroaffinity maturation of Vitaxin, an αvβ3-specific humanized mAb
Authors: Wu Herren , Beuerlein Gregory , Nie Ying , Smith Heidi , Lee Bruce A. , Hensler Mary , Huse William D. , Watkins Jeffry D.
Published: July 2002

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-αvβ3interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to αvβ3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.
Interacting helical faces of subunits
a
and
c
in the F
1
F
o
ATP synthase of
Escherichia coli
defined by disulfide cross-linking
Authors: Jiang Weiping , Fillingame Robert H.
Published: July 2002

Subunits
a
and
c
of F
o
are thought to cooperatively catalyze proton translocation during ATP synthesis by the
Escherichia coli
F
1
F
o
ATP synthase. Optimizing mutations in subunit
a
at residues A217, I221, and L224 improves the partial function of the
c
A24D/
c
D61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit
a
, which then interacted with the transmembrane helix of subunit
c
anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit
c
and the predicted fourth transmembrane helix of subunit
a
. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)
2
SO
4
at 0°, 10°, or 20°C, strong
a–c
dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the
a
S207C/
c
I55C,
a
N214C/
c
A62C,
a
N214C/
c
M65C,
a
I221C/
c
G69C,
a
I223C/
c
L72C,
a
L224C/
c
Y73C, and
a
I225C/
c
Y73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the
a
N214C residue lying close to the
c
A62C and
c
M65C residues in the middle of the membrane. Lesser
a–c
dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit
c
and helix-4 of subunit
a
in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit
c
and helix-4 of subunit
a
pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit
a
and Asp61 in helix-2 of subunit
c
during proton translocation, as has been suggested previously.
Telomere length regulation in mice is linked to a novel chromosome locus
Authors: Zhu Lingxiang , Hathcock Karen S. , Hande Prakash , Lansdorp Peter M. , Seldin Michael F. , Hodes Richard J.
Published: July 2002

Little is known about the mechanisms that regulate species-specific telomere length, particularly in mammalian species. The genetic regulation of telomere length was therefore investigated by using two inter-fertile species of mice, which differ in their telomere length.
Mus musculus
(telomere length >25 kb) and
Mus spretus
(telomere length 5–15 kb) were used to generate F1 crosses and reciprocal backcrosses, which were then analyzed for regulation of telomere length. This analysis indicated that a dominant and trans-acting mechanism exists capable of extensive elongation of telomeres in somatic cells after fusion of parental germline cells with discrepant telomere lengths. A genome wide screen of interspecific crosses, using
M. spretus
as the recurrent parent, identified a 5-centimorgan region on distal chromosome 2 that predominantly controls the observed species-specific telomere length regulation. This locus is distinct from candidate genes encoding known telomere-binding proteins or telomerase components. These results demonstrate that an unidentified gene(s) mapped to distal chromosome 2 regulates telomere length in the mouse.
Growth hormone-releasing hormone antagonist MZ-5-156 inhibits growth of DU-145 human androgen-independent prostate carcinoma in nude mice and suppresses the levels and mRNA expression of insulin-like growth factor II in tumors
Authors: Lamharzi Najib , Schally Andrew V. , Koppán Miklós , Groot Kate
Published: July 2002

Insulin-like growth factors I and II (IGF-I and -II) are potent mitogens for various cancers, including carcinoma of the prostate. In several experimental cancers, treatment with antagonists of growth hormone-releasing hormone (GH-RH) produces a reduction in IGF-I and -II, concomitant to inhibition of tumor growth. To investigate the mechanisms involved, we treated male nude mice bearing xenografts of DU-145 human androgen-independent prostate cancer for 8 weeks with potent GH-RH antagonist MZ-5-156 at a dose of 20 μg/animal s.c. twice a day. Tumor growth, serum and tumor levels of IGF-I and -II, and the mRNA expression of IGF-I and -II in tumors were evaluated. After 8 weeks of therapy, final volume and weight of DU-145 tumors in mice treated with MZ-5-156 were significantly (
P
< 0.01) decreased compared with controls, and serum IGF-I showed a significant reduction. Treatment of nude mice bearing DU-145 xenografts with MZ-5-156 also significantly (
P
< 0.01) diminished by 77% the levels of IGF-II in tumor tissue compared with controls, but did not affect the concentration of IGF-I. Reverse transcription–PCR analyses revealed a high expression of IGF-II mRNA in DU-145 tumors. Treatment with GH-RH antagonist MZ-5-156 decreased the expression of IGF-II mRNA by 58% (
P
< 0.01) as compared with controls. Our work suggests that GH-RH antagonist MZ-5-156 may inhibit the growth of DU-145 human androgen-independent prostate cancers through a reduction in the production and mRNA expression of IGF-II by the tumor tissue. These findings extend our observations on the mechanism of action of GH-RH antagonists and may explain how GH-RH antagonists inhibit tumor growth.
A model to explain the various paradoxes associated with mantle noble gas geochemistry
Authors: Anderson Don L.
Published: July 2002

As a result of an energetic accretion, the Earth is a volatile-poor and strongly differentiated planet. The volatile elements can be accounted for by a late veneer (≈1% of total mass of the Earth). The incompatible elements are strongly concentrated into the exosphere (atmosphere, oceans, sediments, and crust) and upper mantle. Recent geochemical models invoke a large primordial undegassed reservoir with chondritic abundances of uranium and helium, which is clearly at odds with mass and energy balance calculations. The basic assumption behind these models is that excess “primordial”
3
He is responsible for
3
He/
4
He ratios higher than the average for midocean ridge basalts. The evidence however favors depletion of
3
He and excessive depletion of
4
He and, therefore, favors a refractory, residual (low U, Th) source Petrological processes such as melt-crystal and melt-gas separation fractionate helium from U and Th and, with time, generate inhomogeneities in the
3
He/
4
He ratio. A self-consistent model for noble gases involves a gas-poor planet with trapping of CO
2
and noble gases in the shallow mantle. Such trapped gases are released by later tectonic and magmatic processes. Most of the mantle was depleted and degassed during the accretion process. High
3
He/
4
He gases are viewed as products of ancient gas exsolution stored in low U environments, rather than products of primordial reservoirs.
Distribution of genes in the genome of
Arabidopsis thaliana
and its implications for the genome organization of plants
Authors: Barakat Abdelali , Matassi Giorgio , Bernardi Giorgio
Published: July 2002

Previous work has shown that, in the large genomes of three
Gramineae
[rice, maize, and barley: 415, 2,500, and 5,300 megabases (Mb), respectively] most genes are clustered in long DNA segments (collectively called the “gene space”) that represent a small fraction (12–24%) of nuclear DNA, cover a very narrow (0.8–1.6%) GC range, and are separated by vast expanses of gene-empty sequences. In the present work, we have analyzed the small (
ca.
120 Mb) nuclear genome of
Arabidopsis thaliana
and shown that its organization is drastically different from that of the genomes of
Gramineae
. Indeed, (
i
) genes are distributed over about 85% of the main band of DNA in CsCl and cover an 8% GC range; (
ii
) ORFs are fairly evenly distributed in long (>50 kb) sequences from GenBank that amount to about 10 Mb; and (
iii
) the GC levels of protein-coding sequences (and of their third codon positions) are correlated with the GC levels of their flanking sequences. The different pattern of gene distribution of
Arabidopsis
compared with
Gramineae
appears to be because the genomes of the latter comprise (
i
) many large gene-empty regions separating gene clusters and (
ii
) abundant transposons in the intergenic sequences of gene clusters. Both sequences are absent or very scarce in the
Arabidopsis
genome. These observations provide a comparative view of angiosperm genome organization.
Role of the Doc2α–Munc13–1 interaction in the neurotransmitter release process
Authors: Mochida Sumiko , Orita Satoshi , Sakaguchi Gaku , Sasaki Takuya , Takai Yoshimi
Published: July 2002

Doc2α and Munc13–1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca
2+
-dependent neurotransmitter release. Doc2α interacts with Munc13–1 through the N-terminal region of Doc2α (the Mid domain; amino acid residues 13–37). Here we examine whether the interaction between Doc2α and Munc13–1 is required for Ca
2+
-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2α and Munc13–1
in vitro
. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca
2+
concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca
2+
than at 5.1 mM Ca
2+
. These results suggest that the Doc2α–Munc13–1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.
Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites
Authors: Kwok Yan , Zeng Qingping , Hurley Laurence H.
Published: July 2002

Psorospermin is a plant natural product that shows significant
in vivo
activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25-fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase II–DNA complex. Because thermal treatment of (N7 guanine)–DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.
Structure-based assignment of the biochemical function of a hypothetical protein: A test case of structural genomics
Authors: Zarembinski Thomas I. , Hung Li-Wei , Mueller-Dieckmann Hans-Joachim , Kim Kyeong-Kyu , Yokota Hisao , Kim Rosalind , Kim Sung-Hou
Published: July 2002

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms’ predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile,
Methanococcus jannaschii
,
at 1.7-Å resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.
A new Eocene archaeocete (Mammalia, Cetacea) from India and the time of origin of whales
Authors: Bajpai Sunil , Gingerich Philip D.
Published: July 2002

Himalayacetus subathuensis
is a new pakicetid archaeocete from the Subathu Formation of northern India. The type dentary has a small mandibular canal indicating a lack of auditory specializations seen in more advanced cetaceans, and it has
Pakicetus
-like molar teeth suggesting that it fed on fish.
Himalayacetus
is significant because it is the oldest archaeocete known and because it was found in marine strata associated with a marine fauna.
Himalayacetus
extends the fossil record of whales about 3.5 million years back in geological time, to the middle part of the early Eocene [≈53.5 million years ago (Ma)]. Oxygen in the tooth-enamel phosphate has an isotopic composition intermediate between values reported for freshwater and marine archaeocetes, indicating that
Himalayacetus
probably spent some time in both environments. When the temporal range of Archaeoceti is calibrated radiometrically, comparison of likelihoods constrains the time of origin of Archaeoceti and hence Cetacea to about 54–55 Ma (beginning of the Eocene), whereas their divergence from extant Artiodactyla may have been as early as 64–65 Ma (beginning of the Cenozoic).
BRCA1 regulates p53-dependent gene expression
Authors: Ouchi Toru , Monteiro Alvaro N. A. , August Avery , Aaronson Stuart A. , Hanafusa Hidesaburo
Published: July 2002

Mutations in
BRCA1
are present in 45% of families that segregate with susceptibility for breast cancer and in 80–90% of families with both breast and ovarian cancer. Here we report that BRCA1 stimulates artificial and genomic promoter constructs containing p53-responsive elements. This activity of BRCA1 depends on the presence of wild-type p53, which was shown by using mouse fibroblasts expressing temperature-sensitive forms of p53, or p53(+/+) and p53(−/−) fibroblasts obtained from p53 knockout mice. Furthermore, mutant forms of BRCA1 lacking the C-terminal second BRCA1 C-terminal (BRCT) domain showed reduced p53-mediated transcriptional activation. Finally, we found that BRCA1 coimmunoprecipitates with p53,
in vitro
and
in vivo
. These findings suggest a function of BRCA1 as a p53 coactivator.
Hypervariability generated by natural selection in an extracellular complement-inhibiting protein of serotype M1 strains of group AStreptococcus
Authors: Stockbauer Kathryn E. , Grigsby Diana , Pan Xi , Fu Yun-Xin , Mejia Luis M. Perea , Cravioto Alejandro , Musser James M.
Published: July 2002

In many countries, M1 strains of the human pathogenic bacterium group AStreptococcusare the most common serotype recovered from patients with invasive disease episodes. Strains of this serotype express an extracellular protein that inhibits complement [streptococcal inhibitor of complement (Sic)] and is therefore believed to be a virulence factor. Comparative sequence analysis of the 915-bpsicgene in 165 M1 organisms recovered from diverse localities and infection types identified 62 alleles. Inasmuch as multilocus enzyme electrophoresis and pulsed-field gel electrophoresis previously showed that most M1 organisms represent a distinct streptococcal clone, the extent ofsicgene polymorphism was unexpected. The level of polymorphism greatly exceeds that recorded for all other genes examined in serotype M1 strains. All insertions and deletions are in frame, and virtually all nucleotide substitutions alter the amino acid sequence of the Sic protein. These molecular features indicate that structural change in Sic is mediated by natural selection. Study of 70 strains recovered from two temporally distinct epidemics of streptococcal infections in the former East Germany found little sharing of Sic variants among strains recovered in the different time periods. Taken together, the data indicate thatsicis a uniquely variable gene and provide insight into a potential molecular mechanism contributing to fluctuations in streptococcal disease frequency and severity.
In vivo
phosphorylation of the epithelial sodium channel
Authors: Shimkets Richard A. , Lifton Richard , Canessa Cecilia M.
Published: July 2002

The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (α, β, and γ) of ENaC. We found that β and γ, but not the α subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the β and γ subunits in their carboxyl termini, but none of these agents induced
de novo
phosphorylation of α subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the β and γ subunits.
Submicrosecond pacemaker precision is behaviorally modulated: The gymnotiform electromotor pathway
Authors: Moortgat Katherine T. , Keller Clifford H. , Bullock Theodore H. , Sejnowski Terrence J.
Published: July 2002

What are the limits and modulators of neural precision? We address this question in the most regular biological oscillator known, the electric organ command nucleus in the brainstem of wave-type gymnotiform fish. These fish produce an oscillating electric field, the electric organ discharge (EOD), used in electrolocation and communication. We show here that the EOD precision, measured by the coefficient of variation (CV = SD/mean period) is as low as 2 × 10
−4
in five species representing three families that range widely in species and individual mean EOD frequencies (70–1,250 Hz). Intracellular recording in the pacemaker nucleus (Pn), which commands the EOD cycle by cycle, revealed that individual Pn neurons of the same species also display an extremely low CV (CV = 6 × 10
−4
, 0.8 μs SD). Although the EOD CV can remain at its minimum for hours, it varies with novel environmental conditions, during communication, and spontaneously. Spontaneous changes occur as abrupt steps (250 ms), oscillations (3–5 Hz), or slow ramps (10–30 s). Several findings suggest that these changes are under active control and depend on behavioral state: mean EOD frequency and CV can change independently; CV often decreases in response to behavioral stimuli; and lesions of one of the two inputs to the Pn had more influence on CV than lesions of the other input.