bluuebunny/crossref_metadata_2025 huggingface dataset:
Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat.
Authors: Saatman K E , Murai H , Bartus R T , Smith D H , Hayward N J , Perri B R , McIntosh T K
Published: July 2002

Marked increases in intracellular calcium may play a role in mediating cellular dysfunction and death following central nervous system trauma, in part through the activation of the calcium-dependent neutral protease calpain. In this study, we evaluated the effect of the calpain inhibitor AK295 [Z-Leu-aminobutyric acid-CONH(CH2)3-morpholine] on cognitive and motor deficits following lateral fluid percussion brain injury in rats. Before injury, male Sprague-Dawley rats (350-425 g) were trained to perform a beam-walking task and to learn a cognitive test using a Morris water maze paradigm. Animals were subjected to fluid percussion injury (2.2-2.4 atm; 1 atm = 101.3 kPa) and, beginning at 15 min postinjury, received a continuous intraarterial infusion of AK295 (120-140 mg/kg, n = 15) or vehicle (n= 16) for 48 hr. Sham (uninjured) animals received either drug (n = 5) or vehicle (n = 10). Animals were evaluated for neurobehavioral motor function at 48 hr and 7 days postinjury and were tested in the Morris water maze to evaluate memory retention at 7 days postinjury. At 48 hr, both vehicle- and AK295-treated injured animals showed significant neuromotor deficits (P< 0.005). At 7 days, injured animals that received vehicle continued to exhibit significant motor dysfunction (P< 0.01). However, brain-injured, AK295-treated animals showed markedly improved motor scores (P<0.02), which were not significantly different from sham (uninjured) animals. Vehicle-treated, injured animals demonstrated a profound cognitive deficit (P< 0.001), which was significantly attenuated by AK295 treatment (P< 0.05). To our knowledge, this study is the first to use a calpain inhibitor following brain trauma and suggests that calpain plays a role in the posttraumatic events underlying memory and neuromotor dysfunction.
Photochemical electron injection into redox-active proteins
Authors: Brzezinski Peter , Wilson Michael T.
Published: July 2002

A new method is presented that makes it possible to inject electrons rapidly into redox-active proteins by means of a short light flash. Reduced carboxymethylated cytochrome
c
(CmCyt
c
) with carbon monoxide bound to the heme iron is mixed with the oxidized acceptor protein. Upon rapid photodissociation of CO the apparent redox potential of CmCyt
c
drops, resulting in electron transfer to the electron acceptor. In this study we have used mitochondrial cytochrome
c
oxidase as the acceptor protein, but the method also can be used to investigate electron transfer to other proteins that can interact with cytochrome
c
. In principle, it can be used with any redox protein into which a CO binding site at the heme iron can be engineered.
Conditionally replicating mycobacteriophages: A system for transposon delivery to
Mycobacterium tuberculosis
Authors: Bardarov Stoyan , Kriakov Jordan , Carriere Christian , Yu Shengwei , Vaamonde Carlos , McAdam Ruth A. , Bloom Barry R. , Hatfull Graham F. , Jacobs William R.
Published: July 2002

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of
Mycobacterium tuberculosis
has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an
Escherichia coli
cosmid vector containing either a mini-Tn
10
(kan) or Tn
5367
inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (TM4) or 38.5°C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn
10
(kan) occurred in a site-specific fashion in
M. smegmatis
whereas Tn
5367
transposed apparently randomly in
M. phlei
, Bacille Calmette–Guérin (BCG), and
M. tuberculosis
. Sequence analysis of the
M. tuberculosis
and BCG chromosomal regions adjacent to Tn
5367
insertions, in combination with
M. tuberculosis
genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of
M. phlei
, BCG, and
M. tuberculosis
.
Cloning of the cDNA for the TATA-binding protein-associated factor
II
170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and
Drosophila
Authors: van der Knaap Jan A. , Borst Jan Willem , van der Vliet Peter C. , Gentz Reiner , Timmers H. Th. Marc
Published: July 2002

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAF
II
170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAF
II
170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and
Drosophila
moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.
Identification of functional domains on human interleukin 10
Authors: Gesser Borbala , Leffers Henrik , Jinquan Tan , Vestergaard Christian , Kirstein Nicka , Sindet-Pedersen Steen , Jensen Steen Lindkær , Thestrup-Pedersen Kristian , Larsen Christian Grønhøj
Published: July 2002

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytesin vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.
Persistence in muscle of an adenoviral vector that lacks all viral genes
Authors: Chen Hsiao-Huei , Mack Lisa M. , Kelly Robert , Ontell Marcia , Kochanek Stefan , Clemens Paula R.
Published: July 2002

Genetic correction of inherited muscle diseases, such as Duchenne muscular dystrophy, will require long term expression of the recombinant protein following gene transfer. We have shown previously that a new adenoviral vector that lacks all viral genes expressed both full-length dystrophin and β-galactosidase in
mdx
(dystrophin-deficient) mouse muscle. We observed a significant histologic improvement of vector-transduced
mdx
muscle before the eventual loss of vector-encoded transgene expression. In this study, we investigated whether an immunological response against vector-encoded β-galactosidase contributed to the loss of vector expression and affected vector persistence in muscle. Intramuscular vector injection in control normal mice resulted in an early and complete loss of β-galactosidase expression accompanied by predominantly CD4
+
and CD8
+
lymphocytic infiltration and a significant loss of vector DNA. In contrast, intramuscular vector injection in
lacZ
transgenic mice resulted in persistent expression of β-galactosidase for at least 84 days with no evidence of inflammation or significant loss of vector DNA. Our studies demonstrate that, in the absence of an immune response induced by β-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.
Isolation of viable Hodgkin and Reed-Sternberg cells from Hodgkin disease tissues
Authors: Irsch Johannes , Nitsch Silke , Hansmann Martin-Leo , Rajewsky Klaus , Tesch Hans , Diehl Volker , Jox Andrea , Küppers Ralf , Radbruch Andreas
Published: July 2002

Hodgkin disease (HD) is characterized by a small number of malignant Hodgkin and Reed-Sternberg (H/RS) cells among a major population of nonmalignant cells. The analysis of H/RS cells has been hampered by their low frequency and fragility. Here, we describe the isolation of viable H/RS cells from HD affected tissues by high gradient magnetic cell sorting (MACS) according to expression of CD30. The cells were enriched to a purity of up to 50%. H/RS cells were distinguished from other CD30+cells by the expression of CD15, their size and granularity. No CD30/CD15 double-positive cells could be enriched from a lymph node affected by the lymphocyte predominant subtype of HD, activated lymph nodes or peripheral blood of healthy donors. For two cases of HD individual MACS-purified H/RS cells and H/RS cells micromanipulated from tissue sections of the same lymphoma specimens were analyzed for Ig gene rearrangements. In both cases, identical V gene rearrangements were amplified from both sources of H/RS cells, showing that H/RS cells were successfully enriched. Moreover, the finding that in both cases no additional Ig gene rearrangements other than the ones identified in the H/RS cells micromanipulated from tissue sections were amplified from the MACS-purified H/RS cells further supports the monoclonality of these cells throughout the affected lymph nodes. The isolation of viable H/RS cells ex vivo is prerequisite for a direct study of gene expression by those cells and of their interaction with cells in their vicinity.
Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment
Authors: Klug David B. , Carter Carla , Crouch Elizabeth , Roop Dennis , Conti Claudio J. , Richie Ellen R.
Published: July 2002

Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. As expected, cortical and medullary TEC subsets express distinct K expression patterns in the normal thymus. However, we detected two distinct cortical TEC subsets, a major K8+K5−subset and a minor K8+K5+subset, which is highly represented at the cortico-medullary junction. Both cortical TEC subsets are also present in recombination activating gene 1 (RAG-1−/−) and TCRβxδ−/−thymi in which T-cell development is blocked at the CD4−CD8−CD25+CD44−pre-T cell stage. In contrast, K8+K5+TECs predominate in the thymi of human CD3ɛ transgenic mice in which thymocyte development is blocked at an earlier CD4−CD8−CD25−CD44+stage. Transplantation of newborn human CD3ɛ transgenic thymi under the kidney capsule of RAG-1−/−mice results in the emergence of K8+K5−TECs concomitant with the appearance of CD25+thymocytes. Together, the data suggest that cortical TEC development proceeds from a K8+K5+precursor subset to a K8+K5−stage in a differentiation process concomitant with T-cell lineage commitment.
Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK
Authors: Takahashi Chiaki , Sheng Zeqi , Horan Thomas P. , Kitayama Hitoshi , Maki Masatoshi , Hitomi Kiyotaka , Kitaura Yasuyuki , Takai Setsuo , Sasahara Regina M. , Horimoto Aki , Ikawa Yoji , Ratzkin Barry J. , Arakawa Tsutomu , Noda Makoto
Published: July 2002

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-
ras
-transformed NIH 3T3 cells. One such gene,
RECK
, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While
RECK
mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of
RECK
in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.
The origin of the Na-Dene
Authors: Ruhlen Merritt
Published: July 2002

Linguistic evidence indicates that the Yeniseian family of languages, spoken in central Siberia, is most closely related to the Na-Dene family of languages spoken, for the most part, in northwestern North America. This hypothesis locates the source of one of the three migrations responsible for the peopling of the Americas.
The plastid
ndh
genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes
Authors: Sazanov Leonid A. , Burrows Paul A. , Nixon Peter J.
Published: July 2002

The plastid genomes of several plants contain
ndh
genes—homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the
ndh
gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant
Pisum sativum
. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.
Potentials of the National Corn Genome Initiative
Authors: Coe E. H.
Published: July 2002

The present paper summarizes future needs in
information and tools, technology, infrastructure, training, funding,
and bioinformatics, to provide the genomic knowledge and tools for
breeding and biotechnological goals in maize. The National Corn Genome
Initiative (NCGA) has developed through actions taken by the National
Corn Growers Association (NCGA) and participation in a planning process
by institutions, companies, and organizations. At the web address for
the NCGI,
http://www.inverizon.com
/ncgi, are
detailed analyses of goals and costs, impact and value, and strategy
and approaches. The NCGI has also produced an informative and
perceptive video suitable for public groups or schools, about
agricultural contributions to life and the place of maize in these
contributions. High potential can be expected, from cross-application
of knowledge obtained in maize and other cereals. Development of
information and tools for all crops, whether monocots or dicots, will
be gained through an initiative, and each crop will be positioned to
advance with cost-effective parallels, especially for expressed
sequences, markers, and physical mapping.
Production of medakafish chimeras from a stable embryonic stem cell line
Authors: Hong Yunhan , Winkler Christoph , Schartl Manfred
Published: July 2002

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for
in vitro
selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (
Oryzias latipes
) that shows all characteristics of mouse ES cells
in vitro
. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.
Myxococcus
cells respond to elastic forces in their substrate
Authors: Fontes Marta , Kaiser Dale
Published: July 2002

Elasticotaxis describes the ability of
Myxococcus xanthus
cells to sense and to respond to elastic forces within an agar gel on which they rest. Within 5 min of the application of stress, each cell begins to reorient its long axis perpendicular to the stress force. The cells then glide in that direction, and the swarm becomes asymmetric. A quantifiable assay for the strength of elasticotaxis is based on the change in swarm shape from circular to elliptic. By using a collection of isogenic motility mutants, it has been found that the ability to respond to stress in agar depends totally on adventurous (A) motility, but not at all on social (S) motility or on the
frz
genes. In fact, S
−
mutants (which are moving only by means of A motility) respond to the applied stress more strongly than does the wild type, despite the fact that their spreading rates are slower than that of the wt strain. Based on the swarming and elasticotactic phenotypes of isogenic
frizzy
strains that were also defective either in A or S motility,
frz
behaves as if part of the S motility system.
Immediate thalamic sensory plasticity depends on corticothalamic feedback
Authors: Krupa David J. , Ghazanfar Asif A. , Nicolelis Miguel A. L.
Published: July 2002

Multiple neuron ensemble recordings were obtained simultaneously from both the primary somatosensory (SI) cortex and the ventroposterior medial thalamus (VPM) before and during the combined administration of reversible inactivation of the SI cortex and a reversible subcutaneous block of peripheral trigeminal nerve fibers. This procedure was performed to quantify the contribution of descending corticofugal projections on (
i
) the normal organization of thalamic somatosensory receptive fields and (
ii
) the thalamic somatosensory plastic reorganization that immediately follows a peripheral deafferentation. Reversible inactivation of SI cortex resulted in immediate changes in receptive field properties throughout the VPM. Cortical inactivation also significantly reduced but did not completely eliminate the occurrence of VPM receptive field reorganization resulting from the reversible peripheral deafferentation. This result suggests that the thalamic plasticity that is seen immediately after a peripheral deafferentation is dependent upon both descending corticofugal projections and ascending trigeminothalamic projections.
Passage through stationary phase advances replicative aging in
Saccharomyces cerevisiae
Authors: Ashrafi Kaveh , Sinclair David , Gordon Jeffrey I. , Guarente Leonard
Published: July 2002

Saccharomyces cerevisiae
mother cells undergo an aging program that includes morphologic changes, sterility, redistribution of the Sir transcriptional silencing complex from
HM
loci and telomeres to the nucleolus, alterations in nucleolar architecture, and accumulation of extrachromosomal ribosomal DNA circles (ERCs). We report here that cells starved for nutrients during prolonged periods in stationary phase show a decrease in generational lifespan when they reenter the cell cycle. This shortened lifespan is not transmitted to progeny cells, indicating that it is not due to irreversible genetic damage. The decrease in the lifespan is accompanied by all of the changes of accelerated aging with the notable exception that ERC accumulation is not augmented compared with generation-matched, nonstarved cells. These results suggest a number of models, including one in which starvation reveals a component of aging that works in parallel with the accumulation of ERCs. Stationary-phase yeast cells may be a useful system for identifying factors that affect aging in other nondividing eukaryotic cells.
Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic
Salmonella typhimurium
strain
Authors: Mirold Susanne , Rabsch Wolfgang , Rohde Manfred , Stender Silke , Tschäpe Helmut , Rüssmann Holger , Igwe Emeka , Hardt Wolf-Dietrich
Published: July 2002

Salmonella typhimurium
employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few
S. typhimurium
isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEΦ) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different
S. typhimurium
strains. Strikingly, most of the isolates harboring SopEΦ belong to the small group of epidemic strains of
S. typhimurium
that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of
Salmonella
spp. with their hosts.
A nuclear protein tyrosine phosphatase is required for the inactivation of Stat1
Authors: Haspel Richard L. , Darnell James E.
Published: July 2002

The Stat1 activation-inactivation cycle involves phosphorylation of Stat1 in the cytoplasm, translocation to the nucleus, and then a return of the protein to the cytoplasm in a dephosphorylated state. However, the intracellular site of Stat1 dephosphorylation has not been determined. As receptor signaling declines, the flow of activated Stat1 molecules should be to the site of their dephosphorylation. We found that upon receptor-Janus kinase inactivation, either gradual or abruptly induced by staurosporine treatment, the flow of Stat1 was from cytoplasm to the nucleus and the nucleus was the final compartment in which phosphorylated Stat1 was detected. N-terminal mutants of Stat1, previously shown to remain phosphorylated for a longer time than wild-type Stat1, were able to enter the nucleus and were not inactivated in the presence of staurosporine, directly demonstrating that these mutations affect phosphatase access and/or activity during the normal dephosphorylation of Stat1. In the presence of sodium vanadate, a phosphatase inhibitor, phosphorylated Stat1 accumulated in the nucleus as the total amount of Stat1 in the cytoplasm declined to low levels. We conclude that the nucleus is the site of Stat1 inactivation and that dephosphorylation is required for the rapid nuclear export of Stat1.
The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53
Authors: Gobert Céline , Skladanowski Andrzej , Larsen Annette K.
Published: July 2002

DNA topoisomerase I is a nuclear enzyme involved in transcription, recombination, and DNA damage recognition. Previous studies have shown that topoisomerase I interacts directly with the tumor-suppressor protein p53. p53 is a transcription factor that activates certain genes through binding to specific DNA sequences. We now report that topoisomerase I can be stimulated by both latent and activated wild-type p53 as well as by several mutant and truncated p53 proteins
in vitro
, indicating that sequence-specific DNA-binding and stimulation of topoisomerase I are distinct properties of p53. These assays also suggest that the binding site for topoisomerase I on p53 is between amino acids 302 and 321. In living cells, the interaction between p53 and topoisomerase I is strongly dependent on p53 status. In MCF-7 cells, which have wild-type p53, the association between the two proteins is tightly regulated in a spatial and temporal manner and takes place only during brief periods of genotoxic stress. In marked contrast, the two proteins are constitutively associated in HT-29 cells, which have mutant p53. These findings have important implications for both cellular stress response and genomic stability, given the ability of topoisomerase I to recognize DNA lesions as well as to cause illegitimate recombination.
Kinetic evidence for an on-pathway intermediate in the folding of cytochrome
c
Authors: Bai Yawen
Published: July 2002

An early folding event of cytochrome
c
populates a helix-containing intermediate (I
NC
) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U ↔ I
NC
↔ N, or whether the I must first move back through the U and then fold to the N through some alternative path (I
NC
↔ U ↔ N). By means of a kinetic test, it is shown here that the cytochrome
c
I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I ↔ U ↔ N option.
Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C
Authors: Staxén Irina , Pical Christophe , Montgomery Lucy T. , Gray Julie E. , Hetherington Alistair M. , McAinsh Martin R.
Published: July 2002

Oscillations in cytosolic free Ca
2+
concentration
([Ca
2+
]
cyt
) are an important component of
Ca
2+
-based signal transduction pathways. This fact has led
us to investigate whether oscillations in
[Ca
2+
]
cyt
are involved in the response of
stomatal guard cells to the plant hormone abscisic acid (ABA). We show
that ABA induces oscillations in guard-cell
[Ca
2+
]
cyt
. The pattern of the oscillations
depended on the ABA concentration and correlated with the final
stomatal aperture. We examined the mechanism by which ABA generates
oscillations in guard-cell [Ca
2+
]
cyt
by using
1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione
(U-73122), an inhibitor of phosphoinositide-specific phospholipase C
(PI-PLC)-dependent processes in animals. U-73122 inhibited the
hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant
PI-PLC, isolated from a guard-cell-enriched cDNA library, in a
dose-dependent manner. This result confirms that U-73122 is an
inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced
oscillations in [Ca
2+
]
cyt
and stomatal
closure. In contrast, U-73122 did not inhibit external
Ca
2+
-induced oscillations in guard-cell
[Ca
2+
]
cyt
and stomatal closure. Furthermore,
there was no effect of the inactive analogue
1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-2,5-pyrrolidinedione
on recombinant PI-PLC activity or ABA-induced and external
Ca
2+
-induced oscillations in
[Ca
2+
]
cyt
and stomatal closure. This lack of
effect suggests that the effects of U-73122 in guard cells are the
result of inhibition of PI-PLC and not a consequence of nonspecific
effects. Taken together, our data suggest a role for PI-PLC in the
generation of ABA-induced oscillations in
[Ca
2+
]
cyt
and point toward the involvement of
oscillations in [Ca
2+
]
cyt
in the maintenance
of stomatal aperture by ABA.
Oceanic minerals: Their origin, nature of their environment, and significance
Authors: Kastner Miriam
Published: July 2002

The chemical and isotopic compositions of oceanic biogenic and authigenic minerals contain invaluable information on the evolution of seawater, hence on the history of interaction between tectonics, climate, ocean circulation, and the evolution of life. Important advances and greater understanding of (a) key minor and trace element cycles with various residence times, (b) isotopic sources and sinks and fractionation behaviors, and (c) potential diagenetic problems, as well as developments in high-precision instrumentation, recently have been achieved. These advances provided new compelling evidence that neither gradualism nor uniformitarianism can explain many of the new important discoveries obtained from the chemistry and isotopic compositions of oceanic minerals. Presently, the best-developed geochemical proxies in biogenic carbonates are18O/16O and Sr/Ca ratios (possibly Mg/Ca) for temperature;87Sr/86Sr for input sources, Cd/Ca and Ba/Ca ratios for phosphate and alkalinity concentrations, respectively, thus also for ocean circulation;13C/12C for ocean productivity; B isotopes for seawater pH;, U, Th isotopes, and14C for dating; and Sr and Mn concentrations for diagenesis. The oceanic authigenic minerals most widely used for chemical paleoceanography are barite, evaporite sulfates, and hydrogenous ferromanganese nodules. Marine barite is an effective alternative monitor of seawater87Sr/86Sr, especially where carbonates are diagenetically altered or absent. It also provides a high-resolution record of seawater sulfate S isotopes, (evaporite sulfates only carry an episodic record), with new insights on factors affecting the S and C cycles and atmospheric oxygen. High-resolution studies of Sr, Nd, and Pb isotopes of well-dated ferromanganese nodules contain invaluable records on climate driven changes in oceanic circulation.
The anterior determinant
bicoid
of
Drosophila
is a derived
Hox
class 3 gene
Authors: Stauber Michael , Jäckle Herbert , Schmidt-Ott Urs
Published: July 2002

The
Drosophila
gene
bicoid
functions as the anterior body pattern organizer of
Drosophila
. Embryos lacking maternally expressed
bicoid
fail to develop anterior segments including head and thorax. In wild-type eggs,
bicoid
mRNA is localized in the anterior pole region and the
bicoid
protein forms an anterior-to-posterior concentration gradient.
bicoid
activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal
caudal
mRNA in the anterior region of the embryo.
caudal
genes as well as other homeobox genes or members of the
Drosophila
segmentation gene cascade have been found to be conserved in animal evolution. In contrast,
bicoid
homologs have been identified only in close relatives of the schizophoran fly
Drosophila
. This poses the question of how the
bicoid
gene evolved and adopted its unique function in organizing anterior–posterior polarity. We have cloned
bicoid
from a basal cyclorrhaphan fly,
Megaselia abdita
(Phoridae, Aschiza), and show that the gene originated from a recent duplication of the direct homolog of the vertebrate gene
Hox3
, termed
zerknüllt
, which specifies extraembryonic tissues in insects.
Distribution of human herpesvirus-8 latently infected cells in Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma
Authors: Dupin Nicolas , Fisher Cyril , Kellam Paul , Ariad Sam , Tulliez Micheline , Franck Nathalie , van Marck Eric , Salmon Dominique , Gorin Isabelle , Escande Jean-Paul , Weiss Robin A. , Alitalo Kari , Boshoff Chris
Published: July 2002

Human herpesvirus 8 (HHV-8, also called KSHV) is linked to the etiopathogenesis of Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL). The universal presence of HHV-8 in early KS has not yet been shown. We used a mAb (LN53) against latent nuclear antigen-1 (LNA-1) of HHV-8 encoded by ORF73 to study the distribution of the cell types latently infected by HHV-8 in patch, plaque, and nodular KS, MCD, and PEL. In early KS, HHV-8 is present in <10% of cells forming the walls of ectatic vessels. In nodular KS, HHV-8 is present in cells surrounding slit-like vessels and in >90% of spindle cells, but not in normal vascular endothelium. In addition, HHV-8 colocalizes with vascular endothelial growth factor receptor-3 (VEGFR-3), a marker of lymphatic and precursor endothelium. In early KS lesions, VEGFR-3 is more extensively expressed than LNA-1, indicating that HHV-8 is not inducing the proliferation of VEGFR-3-positive endothelium directly. In MCD, HHV-8 is present in mantle zone large immunoblastic B cells. No staining for LNA-1 is seen in samples from multiple myeloma, prostate cancer, and angiosarcoma, supporting the absence of any etiological link between these diseases and HHV-8.
Coexistence of linear zones and pinwheels within orientation maps in cat visual cortex
Authors: Shmuel Amir , Grinvald Amiram
Published: July 2002

Revealing the layout of cortical maps is important both for understanding the processes involved in their development and for uncovering the mechanisms underlying neural computation. The typical organization of orientation maps in the cat visual cortex is radial; complete orientation cycles are mapped around orientation singularities. In contrast, long linear zones of orientation representation have been detected in the primary visual cortex of the tree shrew. In this study, we searched for the existence of long linear sequences and wide linear zones within orientation preference maps of the cat visual cortex. Optical imaging based on intrinsic signals was used. Long linear sequences and wide linear zones of preferred orientation were occasionally detected along the border between areas 17 and 18, as well as within area 18. Adjacent zones of distinct radial and linear organizations were observed across area 18 of a single hemisphere. However, radial and linear organizations were not necessarily segregated; long (7.5 mm) linear sequences of preferred orientation were found embedded within a typical pinwheel-like organization of orientation. We conclude that, although the radial organization is dominant, perfectly linear organization may develop and perform the processing related to orientation in the cat visual cortex.
Osmoregulated ABC-transport system of
Lactococcus lactis
senses water stress via changes in the physical state of the membrane
Authors: van der Heide Tiemen , Poolman Bert
Published: July 2002

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein–lipid interactions.
Analysis of topoisomerase function in bacterial replication fork movement: Use of DNA microarrays
Authors: Khodursky Arkady B. , Peter Brian J. , Schmid Molly B. , DeRisi Joseph , Botstein David , Brown Patrick O. , Cozzarelli Nicholas R.
Published: July 2002

We used DNA microarrays of the
Escherichia coli
genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA
in vivo
, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.
Small molecule developmental screens reveal the logic and timing of vertebrate development
Authors: Peterson Randall T. , Link Brian A. , Dowling John E. , Schreiber Stuart L.
Published: July 2002

Much has been learned about vertebrate development by random
mutagenesis followed by phenotypic screening and by targeted gene
disruption followed by phenotypic analysis in model organisms. Because
the timing of many developmental events is critical, it would be useful
to have temporal control over modulation of gene function, a luxury
frequently not possible with genetic mutants. Here, we demonstrate that
small molecules capable of conditional gene product modulation can be
identified through developmental screens in zebrafish. We have
identified several small molecules that specifically modulate various
aspects of vertebrate ontogeny, including development of the central
nervous system, the cardiovascular system, the neural crest, and the
ear. Several of the small molecules identified allowed us to dissect
the logic of melanocyte and otolith development and to identify
critical periods for these events. Small molecules identified in this
way offer potential to dissect further these and other developmental
processes and to identify novel genes involved in vertebrate
development.
Activin receptor-like kinase 1 modulates transforming growth factor-β1 signaling in the regulation of angiogenesis
Authors: Oh S. Paul , Seki Tsugio , Goss Kendrick A. , Imamura Takeshi , Yi Youngsuk , Donahoe Patricia K. , Li Li , Miyazono Kohei , ten Dijke Peter , Kim Seongjin , Li En
Published: July 2002

The activin receptor-like kinase 1 (ALK1) is a type I receptor for transforming growth factor-β (TGF-β) family proteins. Expression of ALK1 in blood vessels and mutations of the
ALK1
gene in human type II hereditary hemorrhagic telangiectasia patients suggest that ALK1 may have an important role during vascular development. To define the function of ALK1 during development, we inactivated the
ALK1
gene in mice by gene targeting. The
ALK1
homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels. These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by deficient differentiation and recruitment of vascular smooth muscle cells. The blood vessel defects in ALK1-deficient mice are reminiscent of mice lacking TGF-β1, TGF-β type II receptor (TβR-II), or endoglin, suggesting that ALK1 may mediate TGF-β1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-β1 and TβR-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-β1-dependent transcriptional activation mediated by the known TGF-β1 type I receptor, ALK5. Taken together, our results suggest that the balance between the ALK1 and ALK5 signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.
Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2
Authors: Wu Xinyi , Hepner Karin , Castelino-Prabhu Shobha , Do Duc , Kaye Marc B. , Yuan Xiu-Juan , Wood Jonathan , Ross Christopher , Sawyers Charles L. , Whang Young E.
Published: July 2002

PTEN
is a tumor suppressor gene mutated in human cancers. Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif. Using the yeast two-hybrid system, we isolated a membrane-associated guanylate kinase family protein with multiple PDZ domains [AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)]. MAGI-2 contains eight potential protein–protein interaction domains and is localized to tight junctions in the membrane of epithelial cells. PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2. MAGI-2 enhances the ability of PTEN to suppress Akt activation. Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein. We propose that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane.
Genetic evidence that the bacteriophage φX174 lysis protein inhibits cell wall synthesis
Authors: Bernhardt Thomas G. , Roof William D. , Young Ry
Published: July 2002

Protein E, a 91-residue membrane protein of φX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene
slyD
(sensitivity to lysis) that block the lytic effects of E. The role of
slyD
, which encodes a FK506 binding protein-type peptidyl-prolyl
cis-trans
isomerase, is not fully understood. However, E mutants referred to as
Epos
(plates on
slyD
) lack a
slyD
requirement, indicating that
slyD
is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of
Epos
. In this study, we describe the isolation of dominant mutations in the essential host gene
mraY
that result in a general lysis-defective phenotype.
mraY
encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.
Regulation of CD40 function by its isoforms generated through alternative splicing
Authors: Tone Masahide , Tone Yukiko , Fairchild Paul J. , Wykes Michelle , Waldmann Herman
Published: July 2002

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-κB, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.
Archaeological evidence of teosinte domestication from Guilá Naquitz, Oaxaca
Authors: Benz Bruce F.
Published: July 2002

Analysis of the three most ancient
Zea mays
inflorescence fragments from Guilá Naquitz, Oaxaca, Mexico shows
they did not disarticulate naturally, indicating that agricultural
selection of domesticated teosinte was underway by 5,400
14
C years before the present (about 4,200 dendrocalibrated
years B.C.). The cooccurrence of two-ranked specimens with two rows and
four rows of grain and numerous additional morphological
characteristics of these specimens support hypotheses based on
molecular and quantitative genetic analyses that maize evolved from
teosinte. Domestication of the wild ancestor of maize occurred before
the end of the 5th millennium B.C.
Traitement de concepts à connotation émotionnelle positive selon la quantité d'indices de récupération accessibles
Authors: Colombel Fabienne
Published: July 2002

Cette recherche examine la manière dont sont traités les items à connotation émotionnelle positive en mémoire. Pour ce faire, nous proposons de faire varier leur niveau de traitement lors de l'encodage (traitement superficiel ou traitement profond) ainsi que leur type de récupération (rappel libre ou reconnaissance). L'effet de la valence émotionnelle est mis en évidence dans la condition “traitement superficiel—rappel libre”. Les sujets rappellent plus d'items positifs que d'items neutres lorsque peu d'indices de récupération sont accessibles Ce résultat semble confirmé par une analyse qualitative qui ne met en évidence un rappel organisé selon la valence émotionnelle que pour la condition “traitement superficiel”. Les résultats et les implications de cette étude sont discutés en relation avec la théorie de Bower (1981, 1987).