bluuebunny/crossref_metadata_2025 huggingface dataset:
Mice and humans perceive multiharmonic communication sounds in the same way
Authors: Ehret Günter , Riecke Sabine
Published: July 2002

Vowels and voiced consonants of human speech and most mammalian vocalizations consist of harmonically structured sounds. The frequency contours of formants in the sounds determine their spectral shape and timbre and carry, in human speech, important phonetic and prosodic information to be communicated. Steady-state partitions of vowels are discriminated and identified mainly on the basis of harmonics or formants having been resolved by the critical-band filters of the auditory system and then grouped together. Speech-analog processing and perception of vowel-like communication sounds in mammalian vocal repertoires has not been demonstrated so far. Here, we synthesize 11 call models and a tape loop with natural wriggling calls of mouse pups and show that house mice perceive this communication call in the same way as we perceive speech vowels: they need the presence of a minimum number of formants (three formants—in this case, at 3.8 + 7.6 + 11.4 kHz), they resolve formants by the critical-band mechanism, group formants together for call identification, perceive the formant structure rather continuously, may detect the missing fundamental of a harmonic complex, and all of these occur in a natural communication situation without any training or behavioral constraints. Thus, wriggling-call perception in mice is comparable with unconditioned vowel discrimination and perception in prelinguistic human infants and points to evolutionary old rules of handling speech sounds in the human auditory system up to the perceptual level.
Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution
Authors: Allard Julie , Grochulski Pawel , Sygusch Jurgen
Published: July 2002

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the
reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate.
The enzyme is a class I aldolase whose reaction mechanism involves
formation of Schiff base intermediates between Lys-133 and a keto
substrate. A covalent adduct was trapped by flash freezing KDPG
aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH
4.6. Structure determination to 1.95-Å resolution showed that pyruvate
had undergone nucleophilic attack with Lys-133, forming a protonated
carbinolamine intermediate, a functional Schiff base precursor, which
was stabilized by hydrogen bonding with active site residues.
Carbinolamine interaction with Glu-45 indicates general base catalysis
of several rate steps. Stereospecific addition is ensured by aromatic
interaction of Phe-135 with the pyruvate methyl group. In the native
structure, Lys-133 donates all of its hydrogen bonds, indicating the
presence of an ɛ-ammonium salt group. Nucleophilic activation is
postulated to occur by proton transfer in the monoprotonated
zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic
pair requires prior side chain rearrangement by protonated Lys-133 to
displace a water molecule, hydrogen bonded to the zwitterionic
residues.
Self-organizing super-structures formed from hydrogen-bonded biimidazolate metal complexes
Authors: Tadokoro Makoto , Kanno Hideaki , Kitajima Tadanori , Shimada-Umemoto Hiromi , Nakanishi Noritaka , Isobe Kiyoshi , Nakasuji Kazuhiro
Published: July 2002

Manipulation of molecular crystals formed from self-organization is one of the important methods to develop the new molecular functional materials. In particular, we look upon mutual cohesive interactions of hydrogen bonding and metal coordination as one useful tool to construct the preprogramming superstructures. In this study, five controlled superstructures of a one-dimensional linear chain—a zigzag ribbon, right-handed and left-handed helices, and a two-dimensional honeycomb sheet—-are newly created by using the neutral metal complexes with some 2,2′-biimidazolate mono-anions.
Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif
Authors: Doray Balraj , Bruns Kerry , Ghosh Pradipta , Kornfeld Stuart A.
Published: July 2002

The GGAs (
G
olgi-localizing,
γ
-adaptin ear homology domain,
A
RF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (
V
PS-27,
H
rs, and
S
TAM) domain followed by a GAT (
G
GA
a
nd
T
OM1) domain, a connecting hinge segment, and a C-terminal GAE (
γ
-
a
daptin
e
ar) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in
in vitro
assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.
Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor α-induced cell death in fibroblasts
Authors: Morita Yoshiaki , Naka Tetsuji , Kawazoe Yoshinori , Fujimoto Minoru , Narazaki Masashi , Nakagawa Reiko , Fukuyama Hidehiro , Nagata Shigekazu , Kishimoto Tadamitsu
Published: July 2002

Signal transducers and activators of transcription (STAT)-induced STAT inhibitor-1 [SSI-1; also known as suppressor of cytokine signaling-1 (SOCS-1)] was identified as a negative feedback regulator of Janus kinase-STAT signaling. We previously generated mice lacking theSSI-1gene (SSI-1 −/−) and showed that thymocytes and splenocytes in SSI-1 −/− mice underwent accelerated apoptosis. In this paper, we show that murine embryonic fibroblasts lacking theSSI-1gene are more sensitive than their littermate controls to tumor necrosis factor-α (TNF-α)-induced cell death. In addition, L929 cells forced to express SSI-1 (L929/SSI-1), but not SSI-3 or SOCS-5, are resistant to TNF-α-induced cell death. Furthermore L929/SSI-1 cells treated with TNF-α sustain the activation of p38 mitogen-activated protein (MAP) kinase. In contrast, SSI-1 −/− murine embryonic fibroblasts treated with TNF-α show hardly any activation of p38 MAP kinase. These findings suggest that SSI-1 suppresses TNF-α-induced cell death, which is mediated by p38 MAP kinase signaling.
Applied molecular evolution of
O
6
-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells
Authors: Davis Brian M. , Encell Lance P. , Zielske Steven P. , Christians Fred C. , Liu Lili , Friebert Sarah E. , Loeb Lawrence A. , Gerson Stanton L.
Published: July 2002

Applied molecular evolution is a rapidly developing technology that
can be used to create and identify novel enzymes that nature has not
selected. An important application of this technology is the creation
of highly drug-resistant enzymes for cancer gene therapy. Seventeen
O
6
-alkylguanine-DNA alkyltransferase (AGT)
mutants highly resistant to
O
6
-benzylguanine
(BG) were identified previously by screening 8 million variants, using
genetic complementation in
Escherichia coli
. To examine
the potential of these mutants for use in humans, the sublibrary of AGT
clones was introduced to human hematopoietic cells and stringently
selected for resistance to killing by the combination of BG and
1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between
the mutants in human cells revealed three AGT mutants that conferred
remarkable resistance to the combination of BG and
1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered
significantly more frequently than the others. Upon further analysis,
this mutant displayed a level of BG resistance in human hematopoietic
cells greater than that of any previously reported mutant.
UvrA and UvrB suppress illegitimate recombination: Synergistic action with RecQ helicase
Authors: Hanada Katsuhiro , Iwasaki Mihoko , Ihashi Sonoe , Ikeda Hideo
Published: July 2002

Illegitimate recombination is a major cause of genetic instability in prokaryotes as well as in eukaryotes. This recombination usually occurs at a low frequency, but it is greatly enhanced by UV irradiation or other environmental stresses. DNA damages produced by these environmental stresses are thought to induce DNA double-strand breaks, leading to illegitimate recombination. In this paper we show that UV-induced illegitimate recombination is enhanced by mutations of nucleotide excision repair genes,
uvrA
or
uvrB
, and partially by
uvrC
mutation, but not by
uvrD
mutation. Unexpectedly, the recombination was enhanced by the
uvrA uvrB
double mutation even without UV irradiation, but the
uvrB uvrC
double mutation has not shown this effect, suggesting that illegitimate recombination is mostly suppressed by UvrA and UvrB. Moreover, illegitimate recombination was synergistically enhanced by the
recQ uvrA
double mutation. In addition, overproduction of the UvrA protein suppressed the hyperrecombination phenotype of the
recQ
or
uvrB
mutant, but it did not affect the UV-sensitive phenotype of the
uvrB
mutant. We concluded that the UvrAB complex suppresses illegitimate recombination in a pathway shared with RecQ helicase. In addition, UvrA protein alone can suppress illegitimate recombination in the pathway, in which RecQ helicase and UvrAB complex work. Possible functions of the proteins involved in these pathways are also discussed.
Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons
Authors: Sawamoto Kazunobu , Nakao Naoyuki , Kobayashi Kazuto , Matsushita Natsuki , Takahashi Hiroshi , Kakishita Koji , Yamamoto Atsuyo , Yoshizaki Takahito , Terashima Toshio , Murakami Fujio , Itakura Toru , Okano Hideyuki
Published: July 2002

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitoredin vitrowith brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP+cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP+cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.
Minimal exclusion of plasma membrane proteins during retroviral envelope formation
Authors: Hammarstedt Maria , Wallengren Kristina , Pedersen Ketil Winther , Roos Norbert , Garoff Henrik
Published: July 2002

The retrovirus forms its envelope by budding at the plasma membrane (PM). This process is primarily driven by its cytoplasmic core-precursor protein, Gag, as shown by the efficient formation of virus-like Gag particles in the absence of its envelope protein, Env. Most interestingly, several studies have demonstrated incorporation of various PM proteins into retrovirus, but the underlying mechanism of this phenomenon has remained elusive. We have purified Moloney murine leukemia virus Gag particles by sedimentation in an iodixanol gradient and donor PMs by flotation in a sucrose gradient and compared their protein compositions at equal lipid basis. We found that most PM proteins are present at similar density in both membranes. The inclusion of PM proteins was unaffected by incorporation of Env protein into the envelope of the Gag particles and whether these were produced at high or low level in the cells. These findings indicate that most PM proteins become incorporated into the retrovirus envelope without significant sorting. This feature of retrovirus assembly should be considered when studying retrovirus functions and developing retrovirus vectors.
Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome
c
oxidase
Authors: Shiva Sruti , Brookes Paul S. , Patel Rakesh P. , Anderson Peter G. , Darley-Usmar Victor M.
Published: July 2002

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome
c
oxidase. Although it is known that inhibition of cytochrome
c
oxidase by NO is competitive with O
2
, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O
2
partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O
2
tensions can vary widely, with NO having a greater inhibitory effect at low O
2
tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O
2
dependent. This consumption modulates inhibition of cytochrome
c
oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.
Binding and disruption of phospholipid bilayers by supramolecular RNA complexes
Authors: Vlassov Alexandre , Khvorova Anastasia , Yarus Michael
Published: July 2002

In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg
2+
and Ca
2+
concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membrane-active end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.
Panhandle PCR for cDNA: A rapid method for isolation of
MLL
fusion transcripts involving unknown partner genes
Authors: Megonigal Maureen D. , Rappaport Eric F. , Wilson Robert B. , Jones Douglas H. , Whitlock James A. , Ortega Jorge A. , Slater Diana J. , Nowell Peter C. , Felix Carolyn A.
Published: July 2002

Identifying translocations of the
MLL
gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of
MLL
make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5′
MLL
breakpoint cluster region at the 5′ ends and random hexamers at the 3′ ends, known
MLL
sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of
MLL
primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal
MLL
allele in the cell line MV4–11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of
MLL
with
AF-10
in one case and a fusion of
MLL
with
ELL
in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of
MLL
with a variety of partner genes. cDNA panhandle PCR is useful for identifying
MLL
translocations and determining unknown partner sequences in the fusion transcripts.
A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells
Authors: Su Zao-zhong , Lebedeva Irina V. , Gopalkrishnan Rahul V. , Goldstein Neil I. , Stein C. A. , Reed John C. , Dent Paul , Fisher Paul B.
Published: July 2002

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (
mda-
7). Ectopic expression of
mda-
7 when using a recombinant adenovirus, Ad.
mda-
7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of
mda-
7, pancreatic carcinoma cells are remarkably refractory to Ad.
mda
-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.
mda-
7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-
ras
pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in
MDA
-7 and
BAX
proteins and increases in the ratio of
BAX
to
BCL-
2 proteins. Moreover, transfection of mutant K-
ras
pancreatic carcinoma cells with an antisense K-
ras
expression vector and infection with Ad.
mda-
7 inhibits colony formation
in vitro
and tumorigenesis
in vivo
in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.
Short-cycle structured intermittent treatment of chronic HIV infection with highly active antiretroviral therapy: Effects on virologic, immunologic, and toxicity parameters
Authors: Dybul Mark , Chun Tae-Wook , Yoder Christian , Hidalgo Bertha , Belson Michael , Hertogs Kurt , Larder Brendan , Dewar Robin L. , Fox Cecil H. , Hallahan Claire W. , Justement J. Shawn , Migueles Stephen A. , Metcalf Julia A. , Davey Richard T. , Daucher Marybeth , Pandya Punita , Baseler Michael , Ward Douglas J. , Fauci Anthony S.
Published: July 2002

Although continuous highly active antiretroviral therapy
(HAART) is effective for many HIV-infected patients, it can be toxic
and prohibitive in cost. By decreasing the total amount of time
patients receive medications, intermittent HAART could reduce toxicity
and cost. Therefore, we initiated a pilot study in which 10
HIV-infected individuals receiving effective therapy that resulted in
levels of HIV RNA <50 copies per ml of plasma and CD4
+
T
cell counts >300 cells per mm
3
of whole blood received
repeated cycles of 7 days on HAART followed by 7 days off of HAART.
Patients maintained suppression of plasma viremia for 32–68 weeks.
There was no significant increase in HIV proviral DNA or
replication-competent HIV in peripheral CD4
+
T cells or HIV
RNA in peripheral blood or lymph node mononuclear cells. There was no
significant change in CD4
+
T cell counts, no significant
increase in CD4
+
or CD8
+
T cells expressing
activation markers or producing IFN-γ in response to HIV, no increase
in CD4
+
T cell proliferation to p24 antigen, and no
evidence for the development of resistance to HAART medications. There
was a significant decrease in serum cholesterol and triglyceride
levels. Thus, in this proof-of-concept study, short-cycle intermittent
HAART maintained suppression of plasma viremia as well as HIV
replication in reservoir sites while preserving CD4
+
T cell
counts. In addition, there was a decrease in serum cholesterol and
triglyceride levels. Intermittent therapy may be an important strategy
to reduce cost and toxicity for HIV-infected individuals.
Functional expression of mouse Mdr1 in an outer membrane permeability mutant of Escherichia coli.
Authors: Béjà O , Bibi E
Published: July 2002

Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a "leaky" (containing a permeable outer membrane) strain of E. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.
PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.
Authors: Shi J , Ross C R , Leto T L , Blecha F
Published: July 2002

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.
Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.
Authors: He X S , Chen H S , Chu K , Rivkina M , Robinson W S
Published: July 2002

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.
The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.
Authors: Niedermann G , King G , Butz S , Birsner U , Grimm R , Shabanowitz J , Hunt D F , Eichmann K
Published: July 2002

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.
Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).
Authors: Schultz D J , Cahoon E B , Shanklin J , Craig R , Cox-Foster D L , Mumma R O , Medford J I
Published: July 2002

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.
Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.
Authors: Xu R H , Dong Z , Maeno M , Kim J , Suzuki A , Ueno N , Sredni D , Colburn N H , Kung H F
Published: July 2002

Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.
Elongation factor TFIIS contains three structural domains: solution structure of domain II.
Authors: Morin P E , Awrey D E , Edwards A M , Arrowsmith C H
Published: July 2002

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.
beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.
Authors: Gervasoni P , Staudenmann W , James P , Gehrig P , Plückthun A
Published: July 2002

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.
Development of pilus organelle subassemblies
in vitro
depends on chaperone uncapping of a beta zipper
Authors: Bullitt Esther , Jones C. Hal , Striker Robert , Soto Gabriel , Jacob-Dubuisson Françoise , Pinkner Jerome , Wick Mary Jo , Makowski Lee , Hultgren Scott J.
Published: July 2002

The major subassemblies of virulence-associated P pili, the
pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE),
were reconstituted from purified chaperone-subunit complexes
in
vitro
. Subunits are held in assembly-competent conformations in
chaperone-subunit complexes prior to their assembly into mature pili.
The PapD chaperone binds, in part, to a conserved motif present at the
C terminus of the subunits via a beta zippering interaction. Amino acid
residues in this conserved motif were also found to be essential for
subunit–subunit interactions necessary for the formation of pili, thus
revealing a molecular mechanism whereby the PapD chaperone may prevent
premature subunit–subunit interactions in the periplasm. Uncapping of
the chaperone-protected C terminus of PapA and PapE was mimicked
in vitro
by freeze–thaw techniques and resulted in the
formation of pilus rods and tip fibrillae, respectively. A mutation in
the leading edge of the beta zipper of PapA produces pilus rods with an
altered helical symmetry and azimuthal disorder. This change in the
number of subunits per turn of the helix most likely reflects
involvement of the leading edge of the beta zipper in forming a
right-handed helical cylinder. Organelle development is a fundamental
process in all living cells, and these studies shed new light on how
immunoglobulin-like chaperones govern the formation of
virulence-associated organelles in pathogenic bacteria.
Microglia-passaged simian immunodeficiency virus induces neurophysiological abnormalities in monkeys
Authors: Prospéro-García Oscar , Gold Lisa H. , Fox Howard S. , Polis Ilham , Koob George F. , Bloom Floyd E. , Henriksen Steven J.
Published: July 2002

Four rhesus macaques were inoculated intravenously with a cryopreserved stock of microglia obtained from a simian immunodeficiency virus (SIV)-infected rhesus macaque. Before infection, three of the four monkeys were trained and tested daily on a computerized neuropsychological test battery. After SIV infection, behavioral testing continued to monitor deficits associated with disease progression. Five additional age-matched, behaviorally trained monkeys served as controls. Neurophysiological testing for visual and auditory evoked responses was accomplished 37–52 weeks after infection in all monkeys. Subsequently, all four SIV-infected monkeys and one control subject were sacrificed, and samples of brain tissue were taken for pathological analysis. SIV-infected monkeys demonstrated abnormal responses in both auditory and visual evoked responses. In addition, around the time of electrophysiological recording, all three SIV-infected, behaviorally trained monkeys exhibited significant decreases in progressive-ratio performance, reflecting a reduction in reinforcer efficacy. One subject also demonstrated impairments in shifting of attentional set and motor ability at that time. Neuropathological evaluation revealed that all four SIV-infected monkeys exhibited numerous perivascular and parenchymal infiltrating T cells. These findings document that SIV causes electrophysiological, behavioral, and neuropathological sequelae similar to what has been observed in the human neuroAIDS syndrome. Our observations further validate the simian model for the investigation of the pathogenesis of AIDS dementia and for the investigation of drugs with potential therapeutic benefits.
Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.
Authors: Ulisse S , Esslemont G , Baker B S , Krishna V , Chatterjee K , Tata J R
Published: July 2002

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.
Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.
Authors: Gong F C , Giddings T H , Meehl J B , Staehelin L A , Galbraith D W
Published: July 2002

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.
Authors: Wölfl S , Martinez C , Rich A , Majzoub J A
Published: July 2002

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.
Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an S
N
2 displacement reaction
Authors: Lightstone Felice C. , Zheng Ya-Jun , Maulitz Andreas H. , Bruice Thomas C.
Published: July 2002

The semiempirical PM3 method, calibrated against
ab initio
HF/6–31+G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of
Xanthobacter autothropicus
. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (
i
) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO
−
with DCE; (
ii
) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (
iii
) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (
i
) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (
ii
) desolvation; and (
iii
) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.
Delayed loss of cholesterol from a localized lipoprotein depot in apolipoprotein A-I-deficient mice
Authors: Stein Olga , Dabach Yedida , Hollander Gideon , Ben-Naim Mazal , Halperin Gideon , Breslow Jan L. , Stein Yechezkiel
Published: July 2002

The anti-atherogenic role of high density lipoprotein is well known even though the mechanism has not been established. In this study, we have used a novel model system to test whether removal of lipoprotein cholesterol from a localized depot will be affected by apolipoprotein A-I (apo A-I) deficiency. We compared the egress of cholesterol injected in the form of cationized low density lipoprotein into the rectus femoris muscle of apo A-I K-O and control mice. When the injected lipoprotein had been labeled with [
3
H]cholesterol, the
t
½ of labeled cholesterol loss from the muscle was about 4 days in controls and more than 7 days in apo A-I K-O mice. The loss of cholesterol mass had an initial slow (about 4 days) and a later more rapid component; after day 4, the disappearance curves for apo A-I K-O and controls began to diverge, and by day 7, the loss of injected cholesterol was significantly slower in apo A-I K-O than in controls. The injected lipoprotein cholesterol is about 70% in esterified form and undergoes hydrolysis, which by day 4 was similar in control and apo A-I K-O mice. The efflux potential of serum from control and apo A-I K-O mice was studied using media containing 2% native or delipidated serum. A significantly lower efflux of [
3
H]cholesterol from macrophages was found with native and delipidated serum from apo A-I K-O mice. In conclusion, these findings show that lack of apo A-I results in a delay in cholesterol loss from a localized depot
in vivo
and from macrophages in culture. These results provide support for the thesis that anti-atherogenicity of high density lipoprotein is related in part to its role in cholesterol removal.
A factor IX-deficient mouse model for hemophilia B gene therapy
Authors: Wang Lili , Zoppè Monica , Hackeng Tilman M. , Griffin John H. , Lee Kuo-Fen , Verma Inder M.
Published: July 2002

We have generated a mouse where the clotting factor IX (
FIX
) gene has been disrupted by homologous recombination. The
FIX
nullizygous (−/−) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (−/−) was restored by introducing wild-type murine
FIX
gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.