bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivorole of B lymphocytes in somatic transgene immunization
Authors: Xiong Sidong , Gerloni Mara , Zanetti Maurizio
Published: July 2002

Immunity generated byin vivoinoculation of plasmid DNA is a straightforward and potentially valuable new approach to immunization. Little is known about the type of cells involved, the various immunological aspects, and the destiny of the transgene. In this report, we describe a system in which immunity is the result ofin vivotargeting of B lymphocytes. This was accomplished using plasmid DNA encoding an immunoglobulin heavy-chain gene under the control of immunoglobulin promoter and enhancer elements. We show persistence of the transgene in splenic B lymphocytes for at least 3 months, i.e., the average life span of long-lived B lymphocytes in the mouse. The transgene could not be detected in any other lymphoid or nonlymphoid organs over a period of 6 months. We also established that the transgene is integrated in the host DNA. These studies bring new understanding to the events underlying thein vivouse of plasmid DNA. Moreover, the characteristics of this new approach make somatic transgene immunization a model system to study the immunogenicity of endogenous antigens in adult animals.
A screen for fast evolving genes from
Drosophila
Authors: Schmid Karl J. , Tautz Diethard
Published: July 2002

In an attempt to quantify the rates of protein sequence divergence in
Drosophila
, we have devised a screen to differentiate between slow and fast evolving genes. We find that over one-third of randomly drawn cDNAs from a
Drosophila melanogaster
library do not cross-hybridize with
Drosophila virilis
DNA, indicating that they evolve with a very high rate. To determine the evolutionary characteristics of such protein sequences, we sequenced their homologs from a more closely related species (
Drosophila yakuba
). The amino acid substitution rates among these cDNAs are among the fastest known and several are only about 2-fold lower than the corresponding values for silent substitutions. An analysis of within-species polymorphisms for one of these sequences reveals an exceptionally high number of polymorphic amino acid positions, indicating that the protein is not under strong negative selection. We conclude that the
Drosophila
genome harbors a substantial proportion of genes with a very high divergence rate.
The identification of CD4
+
T cell epitopes with dedicated synthetic peptide libraries
Authors: Hiemstra Hoebert S. , Duinkerken Gaby , Benckhuijsen Willemien E. , Amons Reinout , de Vries René R. P. , Roep Bart O. , Drijfhout Jan W.
Published: July 2002

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4
+
T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4
+
T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4
+
T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a
Haemophilus influenzae
TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4
+
T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.
Three Ever Shorter Telomere (
EST
) genes are dispensable for
in vitro
yeast telomerase activity
Authors: Lingner Joachim , Cech Thomas R. , Hughes Timothy R. , Lundblad Victoria
Published: July 2002

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function
in vivo
identified four
EST
(for
e
ver
s
horter
t
elomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the
EST2
gene has recently been shown to encode the catalytic component of telomerase. Using an
in vitro
assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying
est1
-Δ,
est3
-Δ, and
cdc13–2

est

mutations. Therefore, while these three genes are necessary for telomerase function
in vivo
, they do not encode components essential for core catalytic activity. When Est2p, the one
EST
gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.
Ape-like or hominid-like? The positional behavior of
Oreopithecus bambolii
reconsidered
Authors: Köhler Meike , Moyà-Solà Salvador
Published: July 2002

Comparative morphological and functional analyses of the skeletal remains of
Oreopithecus bambolii
, a hominoid from the Miocene Mediterranean island of Tuscany–Sardinia (Italy), provides evidence that bipedal activities made up a significant part of the positional behavior of this primate. The mosaic pattern of its postcranial morphology is to some degree convergent with that of
Australopithecus
and functionally intermediate between apes and early hominids. Some unique traits could have been selected only under insular conditions where the absence of predators and the limitation of trophic resources play a crucial role in mammalian evolution.
Interaction of Gal repressor with inducer and operator: Induction of
gal
transcription from repressor-bound DNA
Authors: Chatterjee Sumana , Zhou Yan-Ning , Roy Siddhartha , Adhya Sankar
Published: July 2002

Gal repressor inhibits transcription from the
gal
promoter (
P
1) when it binds to the cognate operator (
O
E
). The repression is relieved by the presence of the inducer
d
-galactose. Compared with its interaction with free repressor,
d
-galactose binds to the repressor–operator complex with 10-fold reduced affinity as determined by fluorescence enhancement measurements. Thermodynamic analysis and fluorescence anisotropy showed that the stability of the repressor–operator complex is reduced by only 7-fold by the presence of the inducer in the complex. The formation of the inducer–repressor–operator ternary complex has been confirmed by CD spectral analysis. Fluorescence spectroscopy and energy transfer experiments suggest that individual allosteric effects of the two ligands, inducer and operator, on Gal repressor are responsible for the slightly weakened stability of the ternary complex compared with the stability of the inducer–repressor and repressor–operator complexes.
In vitro
transcription results demonstrated full derepression of transcription of the
P
1 promoter under conditions in which the concentrations of the inducer–repressor binary complex are severalfold higher than the dissociation constant of the inducer–repressor–operator ternary complex into inducer–repressor and free DNA. These results strongly suggest that the inducer binding to the repressor–operator complex does not lead to dissociation of the repressor from the operator during transcription induction. Because Gal repressor inhibits transcription by modulating the α subunit of the
P
1-bound RNA polymerase, we conclude that the inducer binding to the operator-bound repressor only allosterically relieves the inhibitory effect of repressor on RNA polymerase without dissociating the repressor from DNA.
Human lymphotoxin mutein lacks hypotensive activity but has higherin vivoantitumor activity than lymphotoxin or tumor necrosis factor
Authors: Taniyama Mitsue , Morita Toshimi , Yamagishi Yasuhiro , Kato Akihisa , Bando Chinami , Okawa Noriyuki , Kaji Akira
Published: July 2002

A serious drawback of tumor necrosis factor α (TNF) as a clinical antitumor agent is that it also has hypotensive activity. To overcome this problem, derivatives of its sister cytokine lymphotoxin (TNF-β or LT) were prepared. One of them, mutein 2 (Mut2) has a deletion of amino acids 1–7 but contains substituted amino acids, Met-Phe-Pro at positions 8–10 of the mature human LT. This mutein has no hypotensive activity at the maximum dose (10 mg/kg) tested on rats. In contrast, a much lower dose (1 mg/kg) of TNF and LT caused a significant blood pressure drop.In vivostudies revealed that Mut2 was more effective than TNF or LT against MethA (a mouse tumor line) as judged by the therapeutic ratio [calculated as LD50(dose that kills 50% of the animals)/ED50(dose that reduces the tumor size by 50%)]. With five other different mouse tumors and two different human tumors, Mut2 was also effective and the effectiveness was comparable or superior to that of TNF or LT. These results suggest the possibility that this derivative may be usable as a clinical antitumor agent without the serious side effects associated with TNF.
The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the
Escherichia coli
DnaJ molecular chaperone
Authors: Kelley William L. , Georgopoulos Costa
Published: July 2002

The N-terminal 70 residue “J-domain” of the
Escherichia coli
DnaJ molecular chaperone is the defining and highly conserved feature of a large protein family. Based upon limited, yet significant, amino acid sequence homology to the J-domain, the DNA encoding the T/t common exon of the simian virus 40 (SV40), JC, or BK polyoma virus T antigen oncoproteins was used to construct J-domain replacement chimeras of the
E. coli
DnaJ chaperone. The virally encoded J-domains successfully substituted for the bacterial counterpart
in vivo
as shown by (
i
) complementation for viability at low and high temperature of a hypersensitive bacterial reporter strain, and (
ii
) the restoration of bacteriophage λ plaque forming ability in the same strain. The amino acid change, H42Q, in the SV40 T/t and the JC virus T/t exon, which is positionally equivalent to the canonical
dnaJ259
H33Q mutation within the
E. coli
J-domain, entirely abolished complementing activity. These results strongly suggest that the heretofore functionally undefined viral T/t common exon represents a
bona fide
J-domain that preserves critical features of the characteristic domain fold essential for J-domain interaction with the ATPase domain of the Hsp70 family. This finding has implications for the regulation of DNA tumor virus T antigens by molecular chaperones.
A negative regulator mediates quorum-sensing control of exopolysaccharide production in
Pantoea stewartii
subsp.
stewartii
Authors: von Bodman Susanne Beck , Majerczak Doris R. , Coplin David L.
Published: July 2002

Classical quorum-sensing (autoinduction) regulation, as exemplified by the
lux
system of
Vibrio fischeri
, requires
N
-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For
Pantoea stewartii
subsp.
stewartii
, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes,
esaI
and
esaR
, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator.
esaI
, Δ
esaR
, and Δ
esaI-esaR
mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an
esaR
mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in
P. s.
subsp.
stewartii
, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed
esaR
strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in
P. s.
subsp.
stewartii
may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.
Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome
in vitro
Authors: Kim Tae Kook , Kim Tae Hoon , Maniatis Tom
Published: July 2002

The transcriptional activity of an
in vitro
assembled human interferon-β gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-κB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly
in vitro
. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-β promoter.
Echo-delay resolution in sonar images of the big brown bat,
Eptesicus
fuscus
Authors: Simmons James A. , Ferragamo Michael J. , Moss Cynthia F.
Published: July 2002

Echolocating big brown bats (
Eptesicus fuscus
) broadcast ultrasonic frequency-modulated (FM) biosonar sounds (20–100 kHz frequencies; 10–50 μs periods) and perceive target range from echo delay. Knowing the acuity for delay resolution is essential to understand how bats process echoes because they perceive target shape and texture from the delay separation of multiple reflections. Bats can separately perceive the delays of two concurrent electronically generated echoes arriving as little as 2 μs apart, thus resolving reflecting points as close together as 0.3 mm in range (two-point threshold). This two-point resolution is roughly five times smaller than the shortest periods in the bat’s sounds. Because the bat’s broadcasts are 2,000–4,500 μs long, the echoes themselves overlap and interfere with each other, to merge together into a single sound whose spectrum is shaped by their mutual interference depending on the size of the time separation. To separately perceive the delays of overlapping echoes, the bat has to recover information about their very small delay separation that was transferred into the spectrum when the two echoes interfered with each other, thus explicitly reconstructing the range profile of targets from the echo spectrum. However, the bat’s 2-μs resolution limit is so short that the available spectral cues are extremely limited. Resolution of delay seems overly sharp just for interception of flying insects, which suggests that the bat’s biosonar images are of higher quality to suit a wider variety of orientation tasks, and that biosonar echo processing is correspondingly more sophisticated than has been suspected.
The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function
Authors: Kask Kalev , Zamanillo Daniel , Rozov Andrei , Burnashev Nail , Sprengel Rolf , Seeburg Peter H.
Published: July 2002

Calcium permeability of
l
-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof
in vivo
that elevated calcium influx through AMPARs need not generate pathophysiological consequences.
The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains
Authors: Lopez-Buesa Pascual , Pfund Christine , Craig Elizabeth A.
Published: July 2002

The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of
Saccharomyces cerevisiae
are functionally distinct. Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties. The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70. Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity. In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K
+
whereas Ssb activity does not. Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a
K
m
and
V
max
for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb. Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb. Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself. The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.
The rice genome project in Japan
Authors: Sasaki Takuji
Published: July 2002

Since 1991, the Rice Genome Research
Program in Japan has carried out rice genomics, such as large-scale
cDNA analysis, construction of a fine-scale restriction fragment length
polymorphism map, and physical mapping of the rice genome with yeast
artificial chromosome clones. These studies have made a great impact on
research into grass genomes and made rice a model plant for other
cereal crop research. Starting in 1998, the Rice Genome Research
Program will step into a new stage of genomics—that of genome
sequencing. This project eventually should reveal all of the genomic
sequence information in the rice plant and be an indispensable aid in
understanding the genomics of other grass species.
The partial tandem duplication of
ALL1
(
MLL
) is consistently generated by
Alu-
mediated homologous recombination in acute myeloid leukemia
Authors: Strout Matthew P. , Marcucci Guido , Bloomfield Clara D. , Caligiuri Michael A.
Published: July 2002

Chromosome abnormalities resulting in gene fusions are commonly associated with acute myeloid leukemia (AML), however, the molecular mechanism(s) responsible for these defects are not well understood. The partial tandem duplication of the
ALL1
(
MLL
) gene is found in patients with AML and trisomy 11 as a sole cytogenetic abnormality and in 11% of patients with AML and normal cytogenetics. This defect results from the genomic fusion of
ALL1
intron 6 or intron 8 to
ALL1
intron 1. Here, we examined the DNA sequence at the genomic fusion in nine cases of AML with a tandem duplication of
ALL1
spanning exons 2–6. Each breakpoint occurred within intron 6 of the
ALL1
breakpoint cluster region and within a discrete 3.8-kb region near the 3′ end of intron 1. In seven cases, a distinct point of fusion of intron 6 with intron 1 could not be identified. Instead, the sequence gradually diverged from an
Alu
element in intron 6 to an
Alu
element in intron 1 through a heteroduplex fusion. Thus, these rearrangements appear to be the result of a recombination event between homologous
Alu
sequences in introns 6 and 1. In two cases, the genomic junction was distinct and involved the fusion of a portion of an
Alu
element in intron 6 with non-
Alu
sequence in intron 1. These data support the hypothesis that a recombination event between homologous
Alu
sequences is responsible for the partial tandem duplication of
ALL1
in the majority of AML cases with this genetic defect. Although
Alu
element-mediated homologous recombination events in germline cells are thought to be responsible for partial gene duplications or deletions in many inherited diseases, this appears to be the first demonstration identifying
Alu
element-mediated recombination as a consistent mechanism for gene rearrangement in somatic tissue.
The PapC usher forms an oligomeric channel: Implications for pilus biogenesis across the outer membrane
Authors: Thanassi David G. , Saulino Evan T. , Lombardo Mary-Jane , Roth Robyn , Heuser John , Hultgren Scott J.
Published: July 2002

Bacterial virulence factors are typically surface-associated or secreted molecules that in Gram-negative bacteria must cross the outer membrane (OM). Protein translocation across the bacterial OM is not well understood. To elucidate this process we studied P pilus biogenesis in
Escherichia coli
. We present high-resolution electron micrographs of the OM usher PapC and show that it forms an oligomeric complex containing a channel approximately 2 nm in diameter. This is large enough to accommodate pilus subunits or the linear tip fibrillum of the pilus but not large enough to accommodate the final 6.8-nm-wide helical pilus rod. We show that P pilus rods can be unraveled into linear fibers by incubation in 50% glycerol. Thus, they are likely to pass through the usher in this unwound form. Packaging of these fibers into their final helical structure would only occur outside the cell, a process that may drive outward growth of the pilus organelles. The usher complex appears to be similar to complexes formed by members of the PulD/pIV family of OM proteins, and thus these two protein families, previously thought to be unrelated, may share structural and functional homologies.
Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria
Authors: Lambert J. David , Moran Nancy A.
Published: July 2002

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15–25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.
A quantitative method for evaluating the stabilities of nucleic acids
Authors: Gelfand Craig A. , Plum G. Eric , Mielewczyk Slawomir , Remeta David P. , Breslauer Kenneth J.
Published: July 2002

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed “reference” duplex (usually a Watson-Crick duplex) and a related “test” duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.
Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice
Authors: Shao Changshun , Deng Li , Henegariu Octavian , Liang Li , Raikwar Nandita , Sahota Amrik , Stambrook Peter J. , Tischfield Jay A.
Published: July 2002

Mice heterozygous at
Aprt
(adenine phosphoribosyltransferase) were used as a model to study
in vivo
loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type
Aprt in vivo
were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2,6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP
r
) fibroblast variants were recovered at a median frequency of 12 × 10
−5
from individual ears from progeny of crosses between mouse strains 129/Sv and C3H/HeJ. The frequency of DAP
r
variants varied greatly among individual ears, suggesting that they preexisted
in vivo
and arose at various times during development
.
Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP
r
variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to
Aprt
did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of
Aprt
LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at
APRT
. Thus, this
Aprt
heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination
in vivo
.
Comparing the hypothalamic and extrahypothalamic actions of endogenous hyperleptinemia
Authors: Wang Zhuo-Wei , Zhou Yan-Ting , Kakuma Tetsuya , Lee Young , Higa Moritake , Kalra Satya P. , Dube Michael G. , Kalra Pushpa S. , Unger Roger H.
Published: July 2002

To determine whether the depletion of body fat caused by adenovirus-induced hyperleptinemia is mediated via the hypothalamus, we used as a “bioassay” for hypothalamic leptin activity the hypothalamic expression of a leptin-regulated peptide, cocaine- and amphetamine-regulated transcript (CART). The validation of this strategy was supported by the demonstration that CART mRNA was profoundly reduced in obese rats with impaired leptin action, whether because of ablation of the ventromedial hypothalamus (VMH) or a loss-of-function mutation in the leptin receptor, as in Zucker diabetic fatty rats. We compared leptin activity in normal rats made hyperleptinemic by adenovirus-leptin treatment (43 ± 9 ng/ml, cerebrospinal fluid leptin 100 pg/ml) with normal rats made hyperleptinemic by a 60% fat intake (19 ± 4 ng/ml, cerebrospinal fluid leptin 69 ± 22 pg/ml). CART was increased 5-fold in the former and 2-fold in the latter, yet in adenovirus-induced hyperleptinemia, body fat had disappeared, whereas in high-fat-fed rats, body fat was abundant. Treatment of the high-fat-fed rats with adenovirus-leptin further increased their hyperleptinemia to 56 ± 6 ng/ml without changing CART mRNA or food intake, indicating that leptin action on hypothalamus had not been increased. Nevertheless, their body fat declined 36%, suggesting that an extrahypothalamic mechanism was responsible. We conclude that in diet-induced obesity body-fat depletion by leptin requires supraphysiologic plasma concentrations that exceed the leptin-transport capacity across the blood–brain barrier.
Analysis of population structure of the chestnut blight fungus based on vegetative incompatibility genotypes
Authors: Milgroom Michael G. , Cortesi Paolo
Published: July 2002

Vegetative incompatibility is a self/nonself-recognition system in fungi that has often been used for describing phenotypic diversity in fungal populations. A common hypothesis is that vegetative incompatibility polymorphisms are maintained by balancing selection. However, understanding the evolutionary significance of vegetative incompatibility and the factors that maintain these polymorphisms has been limited by a lack of knowledge of the underlying genetics of vegetative compatibility (vc) types. Genotypes of 64 vc types, controlled by six unlinked vegetative incompatibility (
vic
) loci, have been identified in the chestnut blight fungus,
Cryphonectria parasitica
. By interpreting vc type survey data in terms of
vic
genotypes, we estimated
vic
-allele frequencies and analyzed the multilocus genetic structure of 13 populations in Europe and 3 populations in the U.S. European populations have less vc type diversity than the US populations because of a combination of lower
vic
-allele diversity and limited recombination. Genotypic diversity of 10 populations in Italy correlated to the abundance of sexual structures; however, significant deviations from random mating suggest that either sexual reproduction may not contribute many offspring in these populations or that
vic
genes (or
vic
genotypes) are under selection. Most
vic
-allele frequencies deviated from 0.5, the equilibrium frequency predicted under frequency-dependent selection, providing no evidence for selection acting on these loci.
Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine
Authors: Balabanli Barbaros , Kamisaki Yoshinori , Martin Emil , Murad Ferid
Published: July 2002

Peroxynitrite-dependent formation of nitrotyrosine has been associated with inactivation of various enzymes and proteins possessing functionally important tyrosines. We have previously reported an enzymatic activity modifying the nitrotyrosine residues in nitrated proteins. Here we are describing a nonenzymatic reduction of nitrotyrosine to aminotyrosine, which depends on heme and thiols. Various heme-containing proteins can mediate the reaction, although the reaction also is catalyzed by heme. The reaction is most effective when vicinal thiols are used as reducing agents, although ascorbic acid also can replace thiols with lesser efficiency. The reaction could be inhibited by (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1, but not other tested NO donors. HPLC with electrochemical detection analysis of the reaction identified aminotyrosine as the only reaction product. The reduction of nitrotyrosine was most effective at a pH close to physiological and was markedly decreased in acidic conditions. Various nitrophenol compounds also were modified in this reaction. Understanding the mechanism of this reaction could help define the enzymatic modification of nitrotyrosine-containing proteins. Furthermore, this also could assist in understanding the role of nitrotyrosine formation and reversal in the regulation of various proteins containing nitrotyrosine. It also could help define the role of nitric oxide and other reactive species in various disease states.
Increased thrombin responsiveness in platelets from mice lacking glycoprotein V
Authors: Ramakrishnan Vanitha , Reeves Peter S. , DeGuzman Francis , Deshpande Usha , Ministri-Madrid Kathleen , DuBridge Robert B. , Phillips David R.
Published: July 2002

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V −/− platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard–Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover,
in vitro
analysis demonstrated that GP V −/− platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V −/− mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors.
Functioning of the
Drosophila
integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by
snf
+
gene dose effects
Authors: Cline Thomas W. , Rudner David Z. , Barbash Daniel A. , Bell Maren , Vutien Roselyne
Published: July 2002

Snf, encoded by
sans fille
, is the Drosophila homolog of mammalian U1A and U2B′′ and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf’s functioning within snRNPs.
Sxl
is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant
Sxl
alleles in an
in vivo
assay, we show that Snf is rate-limiting for
Sxl
autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic
snf
+
dose enhances the positive autoregulatory activity of Sxl for
Sxl
somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a
60
, has no effect. Increased
snf
+
enhances
Sxl
autoregulation even when U1-70k and SF3a
60
are reduced by mutation to levels that, in the case of SF3a
60
, demonstrably interfere with
Sxl
autoregulation. The observation that increased
snf
+
does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a
60
is additional evidence that
snf
+
dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.
The role of presynaptic activity in monocular deprivation: Comparison of homosynaptic and heterosynaptic mechanisms
Authors: Blais Brian S. , Shouval Harel Z. , Cooper Leon N.
Published: July 2002

Although investigations in computational neuroscience have been extensive, the opportunity (that has made such a marked difference in physical sciences) to test detailed and subtle quantitative consequences of a theory against experimental results is rare. In this paper, we outline a testable consequence of two contrasting theories of synaptic plasticity applied to the disconnection in visual cortex of the closed eye in monocular deprivation. This disconnection is sometimes thought to be the consequence of a process that stems from a competition of inputs for a limited resource such as neurotrophin. Such a process leads to what we call spatial competition, or heterosynaptic synaptic modification. A contrasting view—exemplified by the Bienenstock, Cooper, and Munro (BCM) theory—is that patterns of input activity compete in the temporal domain. This temporal competition is homosynaptic and does not require a conserved resource. The two mechanisms, homosynaptic and heterosynaptic, are the distinguishing characteristics of two general classes of learning rules we explore by using a realistic environment composed of natural scenes. These alternative views lead to opposite dependence on the level of presynaptic activity of the rate of disconnection of the closed eye in monocular deprivation. This strong and testable consequence sets the stage for a critical distinguishing experiment. This experiment has been done and supports the second view. These results have important implications for the processes of learning and memory storage in neocortex.
Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum
Authors: Suh Jung-Keun , Poulsen Lawrence L. , Ziegler Daniel M. , Robertus Jon D.
Published: July 2002

The flavin-containing monooxygenase from yeast (yFMO) catalyzes the O
2
- and NADPH-dependent oxidations of biological thiols, including oxidation of glutathione to glutathione disulfide (GSSG). Glutathione and GSSG form the principle redox buffering system in the cell, with the endoplasmic reticulum (ER) being more oxidizing than the cytoplasm. Proper folding of disulfide-bonded proteins in the ER depends on an optimum redox buffer ratio. Here we show that yFMO is localized to the cytoplasmic side of the ER membrane. We used a gene knockout strain and expression vectors to show that yFMO has a major effect on the generation of GSSG transported into the ER. The enzyme is required for the proper folding, in the ER, of test proteins with disulfide bonds, whereas those without disulfide bonds are properly folded independently of yFMO in the ER or in the cytoplasm.
Hypervariable region 3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization: Therapeutic and prophylactic implications
Authors: Wang Wei-Kung , Dudek Tim , Essex Max , Lee Tun-Hou
Published: July 2002

Crystallographic characterization of a ternary complex containing a monomeric gp120 core, parts of CD4, and a mAb, revealed a region that bridges the inner and outer domains of gp120. In a related genetic study, several residues conserved among primate lentiviruses were found to play important roles in CC-chemokine receptor 5 (CCR5) coreceptor utilization, and all but one were mapped to the bridging domain. To reconcile this finding with previous reports that the hypervariable region 3 (V3) of gp120 plays an important role in chemokine coreceptor utilization, elucidating the roles of various V3 residues in this critical part of the HIV type 1 (HIV-1) life cycle is essential. Alanine-scanning mutagenesis was carried out to identify V3 residues critical for CCR5 utilization. Our findings demonstrated that several residues in V3 were critical to CCR5 utilization. Furthermore, these residues included not only those conserved across HIV-1 subtypes, but also those that varied among HIV-1 subtypes. Although the highly conserved V3 residues may represent unique targets for antiviral designs, the involvement of variable residues raises the possibility that antigenic variation in the coreceptor binding domain could further complicate HIV-1 vaccine design.
Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast
Authors: Chang Louise , Gould Kathleen L.
Published: July 2002

A mutation in the
Schizosaccharomyces pombe sid4
+
(
s
eptation
i
nitiation
d
efective) gene was isolated in a screen for mutants defective in cytokinesis. We have cloned
sid4
+
and have found that
sid4
+
encodes a previously unknown 76.4-kDa protein that localizes to the spindle pole body (SPB) throughout the cell cycle. Sid4p is required for SPB localization of key regulators of septation initiation, including the GTPase Spg1p, the protein kinase Cdc7p, and the GTPase-activating protein Byr4p. An N-terminally truncated Sid4p mutant does not localize to SPBs and when overproduced acts as a dominant-negative mutant by titrating endogenous Sid4p and Spg1p from the SPB. Conversely, the Sid4p N-terminal 153 amino acids are sufficient for SPB localization. Biochemical studies demonstrate that Sid4p interacts with itself, and yeast two-hybrid analysis shows that its self-interaction domain lies within the C-terminal half of the protein. Our data indicate that Sid4p SPB localization is a prerequisite for the execution of the Spg1p signaling cascade.
A genetic model defines the importance of the atrial natriuretic peptide receptor (guanylyl cyclase-A) in the regulation of kidney function
Authors: Dubois Susan K. , Kishimoto Ichiro , Lillis Terence O. , Garbers David L.
Published: July 2002

Disruption of the atrial natriuretic peptide (ANP) receptor [guanylyl cyclase-A (GC-A)] gene yields mice with a salt-resistant form of hypertension, raising fundamental questions on the role of ANP in acute regulation of the kidney. Here, we show that water intake, food consumption, stool weight, urine volume, and sodium excretion are not significantly different between wild-type and GC-A null mice on standard rodent chow (0.7% NaCl) or a high-salt diet (8% NaCl). In conscious mice with an indwelling catheter, the infusion of a physiological saline solution containing 4% BSA resulted in a marked natriuresis/diuresis in wild-type mice but no response in GC-A null animals. When physiological saline was given by gavage, however, the kidney response of wild-type and null mice was equivalent, raising the possibility that the gastrointestinal tract can directly regulate kidney function. However, administration of 0.9% saline through an intraperitoneal route also resulted in equal kidney responses in wild-type and null mice. When 0.9% NaCl lacking protein was infused i.v., wild-type and null mice both responded at the kidney level. Thus, GC-A appears dispensable for regulation of sodium/water excretion in response to changes in dietary sodium concentration, but likely becomes critical in volume expansions where the isooncotic pressure remains constant, such as head-out immersion or the initial and correctable stages of congestive heart failure.
Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells
Authors: O'Hare Michael J. , Bond Jacquelyn , Clarke Catherine , Takeuchi Yasuhiro , Atherton Amanda J. , Berry Catherine , Moody John , Silver Andrew R. J. , Davies Derek C. , Alsop Amber E. , Neville A. Munro , Jat Parmjit S.
Published: July 2002

Reports differ as to whether reconstitution of telomerase
activity alone is sufficient for immortalization of different types of
human somatic cells or whether additional activities encoded by other
“immortalizing” genes are also required. Here we show that
ectopic expression of either the catalytic subunit of human telomerase
(hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40
large-tumor antigen alone was not sufficient for immortalization of
freshly isolated normal adult human mammary fibroblasts and endothelial
cells. However, a combination of both genes resulted in the efficient
generation of immortal cell lines irrespective of the order in which
they were introduced or whether they were introduced early or late in
the normal proliferative lifespan of the cultures. The order and timing
of transduction, however, did influence genomic stability. Karyotype
analysis indicated that introduction of both transgenes at early
passage, with hTERT first, yielded diploid cell lines.
Temperature-shift experiments revealed that maintenance of the
immortalized state depended on continued expression of functional
U19tsA58 large-tumor antigen, with hTERT alone unable to maintain
growth at nonpermissive temperatures for U19tsA58 large-tumor
antigen. Such conditional diploid lines may provide a useful
resource for both cell engineering and for studies on immortalization
and
in vitro
transformation.
Oligonucleotide-directed peptide synthesis in a ribosome- and ribozyme-free system
Authors: Tamura Koji , Schimmel Paul
Published: July 2002

Peptide bond formation by the ribosome requires 23S rRNA and its
interaction with the 3′-CCA end of tRNA. To investigate the possible
evolutionary development of the peptidyl transfer reaction, we tried to
obtain peptide bond formation without the ribosome or rRNA simply by
using a piece of tRNA—an aminoacyl-minihelix—mixed with
sequence-specific oligonucleotides that contained puromycin. Peptide
bond formation was detected by gel electrophoresis, TLC analysis, and
mass spectrometry. Peptide synthesis depended on sequence
complementarity between the 3′-CCA sequence of the minihelix and the
puromycin-bearing oligonucleotide. However, proximity of the reacting
species was not by itself sufficient for peptide bond formation. In
addition, imidazole as a catalyst was required. Its role may be similar
to the recently proposed mechanism, wherein A2451 of 23S rRNA works as
a general base. Thus, peptide bond formation can be achieved with a
simple, minimized system that captures the essence of an interaction
seen in the ribosome.
New States, Old Identities? The Czech Republic, Slovakia, and Historical Understandings of Statehood
Authors: Agnew Hugh LeCaine
Published: July 2002

Imagine an intrepid web explorer looking for information on the Czech Republic. He or she might well stumble onto the SunSITE at the Faculty of Mathematics and Physics at Charles University in Prague. When our websurfer clicks on the “Czech Republic” link, the screen displays the image shown in Figure 1.