bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of the
Drosophila
homologue of the
Myb
protooncogene causes genomic instability
Authors: Manak J. Robert , Mitiku Nesanet , Lipsick Joseph S.
Published: July 2002

Vertebrates have three related
Myb
genes. The c-
Myb
protooncogene is required for definitive hematopoiesis in mice and when mutated causes leukemias and lymphomas in birds and mammals. The A-
Myb
gene is required for spermatogenesis and mammary gland proliferation in mice. The ubiquitously expressed B-
Myb
gene is essential for early embryonic development in mice and is directly regulated by the p16/cyclin D/Rb family/E2F pathway along with many critical S-phase genes.
Drosophila
has a single
Myb
gene most closely related to B-
Myb
. We have isolated two late-larval lethal alleles of
Drosophila Myb
. Mutant imaginal discs show an increased number of cells arrested in M phase. Mutant mitotic cells display a variety of abnormalities including spindle defects and increased polyploidy and aneuploidy. Remarkably, some mutant cells have an aberrant S- to M-phase transition in which replicating chromosomes undergo premature histone phosphorylation and chromosomal condensation. These results suggest that the absence of
Drosophila Myb
causes a defect in S phase that may result in M-phase abnormalities. Consistent with a role for
Drosophila Myb
during S phase, we detected Dm-Myb protein in S-phase nuclei of wild-type mitotic cells as well as endocycling cells, which lack both an M phase and
cyclin B
expression. Moreover, we found that the Dm-Myb protein is concentrated in regions of S-phase nuclei that are actively undergoing DNA replication. Together these findings imply that Dm-Myb provides an essential nontranscriptional function during chromosomal replication.
A substance P-opioid chimeric peptide as a unique nontolerance-forming analgesic
Authors: Foran Stacy E. , Carr Daniel B. , Lipkowski Andrzej W. , Maszczynska Iwona , Marchand James E. , Misicka Aleksandra , Beinborn Martin , Kopin Alan S. , Kream Richard M.
Published: July 2002

To elucidate mechanisms of acute and chronic pain, it is important to understand how spinal excitatory systems influence opioid analgesia. The tachykinin substance P (SP) represents the prototypic spinal excitatory peptide neurotransmitter/neuromodulator, acting in concert with endogenous opioid systems to regulate analgesic responses to nociceptive stimuli. We have synthesized and pharmacologically characterized a chimeric peptide containing overlapping NH
2
- and COOH-terminal functional domains of the endogenous opioid endomorphin-2 (EM-2) and the tachykinin SP, respectively. Repeated administration of the chimeric molecule YPFFGLM-NH
2
, designated ESP7, into the rat spinal cord produces opioid-dependent analgesia without loss of potency over 5 days. In contrast, repeated administration of ESP7 with concurrent SP receptor (SPR) blockade results in a progressive loss of analgesic potency, consistent with the development of tolerance. Furthermore, tolerant animals completely regain opioid sensitivity after post hoc administration of ESP7 alone, suggesting that coactivation of SPRs is essential to maintaining opioid responsiveness. Radioligand binding and signaling assays, using recombinant receptors, confirm that ESP7 can coactivate μ-opioid receptors (MOR) and SPRs
in vitro
. We hypothesize that coincidental activation of the MOR- and SPR-expressing systems in the spinal cord mimics an ongoing state of reciprocal excitation and inhibition, which is normally encountered in nociceptive processing. Due to the ability of ESP7 to interact with both MOR and SPRs, it represents a unique prototypic, anti-tolerance-forming analgesic with future therapeutic potential.
Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression
Authors: Shevde Nirupama K. , Bendixen Amy C. , Dienger Krista M. , Pike J. Wesley
Published: July 2002

Loss of ovarian function following menopause results in a substantial increase in bone turnover and a critical imbalance between bone formation and resorption. This imbalance leads to a progressive loss of trabecular bone mass and eventually osteoporosis, in part the result of increased osteoclastogenesis. Enhanced formation of functional osteoclasts appears to be the result of increased elaboration by support cells of osteoclastogenic cytokines such as IL-1, tumor necrosis factor, and IL-6, all of which are negatively regulated by estrogens. We show here that estrogen can suppress receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced differentiation of myelomonocytic precursors into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts through an estrogen receptor-dependent mechanism that does not require mediation by stromal cells. This suppression is dose-dependent, isomer-specific, and reversed by ICI 182780. Furthermore, the bone-sparing analogues tamoxifen and raloxifene mimic estrogen's effects. Estrogen blocks RANKL/M-CSF-induced activator protein-1-dependent transcription, likely through direct regulation of c-Jun activity. This effect is the result of a classical nuclear activity by estrogen receptor to regulate both c-Jun expression and its phosphorylation by c-Jun N-terminal kinase. Our results suggest that estrogen modulates osteoclast formation both by down-regulating the expression of osteoclastogenic cytokines from supportive cells and by directly suppressing RANKL-induced osteoclast differentiation.
Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy
Authors: Fong Lawrence , Hou Yafei , Rivas Alberto , Benike Claudia , Yuen Alan , Fisher George A. , Davis Mark M. , Engleman Edgar G.
Published: July 2002

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-foldin vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+T cells, confirming the role of CD8 T cells in this treatment strategy.
Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness
Authors: Van Hooser J. Preston , Aleman Tomas S. , He Yu-Guang , Cideciyan Artur V. , Kuksa Vladimir , Pittler Steven J. , Stone Edwin M. , Jacobson Samuel G. , Palczewski Krzysztof
Published: July 2002

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis.
Rpe65
-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-
cis
-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.
Interaction of
Pseudomonas aeruginosa
with epithelial cells: Identification of differentially regulated genes by expression microarray analysis of human cDNAs
Authors: Ichikawa Jeffrey K. , Norris Anne , Bangera M. Gita , Geiss Gary K. , van 't Wout Angélique B. , Bumgarner Roger E. , Lory Stephen
Published: July 2002

Pseudomonas aeruginosa
is an opportunistic pathogen that plays a major role in lung function deterioration in cystic fibrosis patients. To identify critical host responses during infection, we have used high-density DNA microarrays, consisting of 1,506 human cDNA clones, to monitor gene expression in the A549 lung pneumocyte cell line during exposure to
P. aeruginosa
. We have identified host genes that are differentially expressed upon infection, several of which require interaction with
P. aeruginosa
and the expression of the major subunit of type IV pili, PilA. Differential expression of genes involved in various cellular functions was identified, and we selected the gene encoding the transcription factor interferon regulatory factor 1 (IRF-1) for further analysis. The levels of the IRF-1 transcript increased 3- to 4-fold in A549 cells after adherence by
P. aeruginosa
. A similar increase of IRF-1 mRNA was observed in A549 cells exposed to wild-type
P. aeruginosa
when compared to an isogenic, nonpiliated strain. However, this difference was abolished when serum was present during the incubation of bacteria. Exposure of A549 cells to purified
P. aeruginosa
lipopolysaccharide did not result in a significant increase in IRF-1 mRNA. Although the
P. aeruginosa
-induced increased IRF-1 expression depends on the presence of bacterial adhesin, our findings do not preclude the possibility that other bacterial products are responsible for IRF-1 activation, which is enhanced by bacterial adherence to cells. These data show that microarray technology can be an important tool for studying the complex interplay between bacterial pathogens and host.
Efficient chromosomal transposition of a
Tc1
/
mariner
- like transposon
Sleeping Beauty
in mice
Authors: Horie Kyoji , Kuroiwa Asato , Ikawa Masahito , Okabe Masaru , Kondoh Gen , Matsuda Yoichi , Takeda Junji
Published: July 2002

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no
a priori
assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a
Tc1
/
mariner
-like transposon,
Sleeping Beauty
, in mice. This system allows germ-line mutagenesis
in vivo
and will facilitate certain aspects of phenotype-driven genetic screening in mice.
A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by geneMAGE-3
Authors: Coulie Pierre G. , Karanikas Vaios , Colau Didier , Lurquin Christophe , Landry Claire , Marchand Marie , Dorval Thierry , Brichard Vincent , Boon Thierry
Published: July 2002

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis afterin vitrorestimulation indicated that about 1/40,000 postimmunization CD8+blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 × 106CD8+lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection.
Molecular mechanism of endothelial growth arrest by laminar shear stress
Authors: Lin Kurt , Hsu Pin-Pin , Chen Benjamin P. , Yuan Suli , Usami Shunichi , Shyy John Y.-J. , Li Yi-Shuan , Chien Shu
Published: July 2002

This study was designed to elucidate the mechanism underlying the inhibition of endothelial cell growth by laminar shear stress. Tumor suppressor gene p53 was increased in bovine aortic endothelial cells subjected to 24 h of laminar shear stress at 3 dynes (1 dyne = 10 μN)/cm
2
or higher, but not at 1.5 dynes/cm
2
. One of the mechanisms of the shear-induced increase in p53 is its stabilization after phosphorylation by c-Jun N-terminal kinase. To investigate the consequence of the shear-induced p53 response, we found that prolonged laminar shear stress caused increases of the growth arrest proteins GADD45 (growth arrest and DNA damage inducible protein 45) and p21
cip1
, as well as a decrease in phosphorylation of the retinoblastoma gene product. Our results suggest that prolonged laminar shear stress causes a sustained p53 activation, which induces the up-regulation of GADD45 and p21
cip1
. The resulting inhibition of cyclin-dependent kinase and hypophosphorylation of retinoblastoma protein lead to endothelial cell cycle arrest. This inhibition of endothelial cell proliferation by laminar shear stress may serve an important homeostatic function by preventing atherogenesis in the straight part of the arterial tree that is constantly subjected to high levels of laminar shearing.
Essential role for p38α mitogen-activated protein kinase in placental angiogenesis
Authors: Mudgett John S. , Ding Jixiang , Guh-Siesel Lucia , Chartrain Nicole A. , Yang Lu , Gopal Shobhna , Shen Michael M.
Published: July 2002

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the
p38
α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in
p38
α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular,
p38
α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore,
p38
α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.
Regulation and localization of tyrosine216phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration
Authors: Bhat Ratan V. , Shanley Jason , Correll Maryann P. , Fieles William E. , Keith Richard A. , Scott Clay W. , Lee Chi-Ming
Published: July 2002

Inactivation of glycogen synthase kinase-3β (GSK3β) by S9phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y216, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y216phosphorylation on GSK3β in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y216, GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9phosphorylation and inactivation of GSK3β but did not affect Y216phosphorylation, suggesting that S9phosphorylation is sufficient to override GSK3β activation by Y216phosphorylation. Under the conditions examined, increased Y216phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y216phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y216-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y216phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y216phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.
Genome sequence of
Halobacterium
species NRC-1
Authors: Ng Wailap Victor , Kennedy Sean P. , Mahairas Gregory G. , Berquist Brian , Pan Min , Shukla Hem Dutt , Lasky Stephen R. , Baliga Nitin S. , Thorsson Vesteinn , Sbrogna Jennifer , Swartzell Steven , Weir Douglas , Hall John , Dahl Timothy A. , Welti Russell , Goo Young Ah , Leithauser Brent , Keller Kim , Cruz Randy , Danson Michael J. , Hough David W. , Maddocks Deborah G. , Jablonski Peter E. , Krebs Mark P. , Angevine Christine M. , Dale Heather , Isenbarger Thomas A. , Peck Ronald F. , Pohlschroder Mechthild , Spudich John L. , Jung Kwang-Hwan , Alam Maqsudul , Freitas Tracey , Hou Shaobin , Daniels Charles J. , Dennis Patrick P. , Omer Arina D. , Ebhardt Holger , Lowe Todd M. , Liang Ping , Riley Monica , Hood Leroy , DasSarma Shiladitya
Published: July 2002

We report the complete sequence of an extreme halophile,
Halobacterium sp.
NRC-1, harboring a dynamic
2,571,010-bp genome containing 91 insertion sequences representing 12
families and organized into a large chromosome and 2 related
minichromosomes. The
Halobacterium
NRC-1 genome codes
for 2,630 predicted proteins, 36% of which are unrelated to any
previously reported. Analysis of the genome sequence shows the presence
of pathways for uptake and utilization of amino acids, active
sodium-proton antiporter and potassium uptake systems, sophisticated
photosensory and signal transduction pathways, and DNA replication,
transcription, and translation systems resembling more complex
eukaryotic organisms. Whole proteome comparisons show the definite
archaeal nature of this halophile with additional similarities to the
Gram-positive
Bacillus subtilis
and other bacteria. The
ease of culturing
Halobacterium
and the availability of
methods for its genetic manipulation in the laboratory, including
construction of gene knockouts and replacements, indicate this
halophile can serve as an excellent model system among the archaea.
Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor
Authors: Simonovic Miljan , Gettins Peter G. W. , Volz Karl
Published: July 2002

Pigment epithelium-derived factor (PEDF), a noninhibitory member of
the serpin superfamily, is the most potent inhibitor of angiogenesis in
the mammalian ocular compartment. It also has neurotrophic activity,
both in the retina and in the central nervous system, and is highly
up-regulated in young versus senescent fibroblasts. To provide a
structural basis for understanding its many biological roles, we have
solved the crystal structure of glycosylated human PEDF to 2.85 Å. The
structure revealed the organization of possible receptor and
heparin-binding sites, and showed that, unlike any other previously
characterized serpin, PEDF has a striking asymmetric charge
distribution that might be of functional importance. These results
provide a starting point for future detailed structure/function
analyses into possible mechanisms of PEDF action that could lead to
development of therapeutics against uncontrolled angiogenesis.
Regulation of NMDA receptors by cyclin-dependent kinase-5
Authors: Li Bing-Sheng , Sun Miao-Kun , Zhang Lei , Takahashi Satoru , Ma Wu , Vinade Lucia , Kulkarni Ashok B. , Brady Roscoe O. , Pant Harish C.
Published: July 2002

Members of theN-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232in vitroand in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.
T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries
Authors: Danner Stefan , Belasco Joel G.
Published: July 2002

RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable
in vitro
genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3′ stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10
7
independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (
K
d
as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B" demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins.
Behavioral insensitivity to restraint stress, absent fear suppression of behavior and impaired spatial learning in transgenic rats with hippocampal neuropeptide Y overexpression
Authors: Thorsell A. , Michalkiewicz M. , Dumont Y. , Quirion R. , Caberlotto L. , Rimondini R. , Mathé A. A. , Heilig M.
Published: July 2002

Exogenous neuropeptide Y (NPY) reduces experimental anxiety in a
wide range of animal models. The generation of an NPY-transgenic rat
has provided a unique model to examine the role of
endogenous NPY in control of stress and anxiety-related
behaviors using paradigms previously used by pharmacological studies.
Locomotor activity and baseline behavior on the elevated plus maze were
normal in transgenic subjects. Two robust phenotypic traits were
observed. (
i
) Transgenic subjects showed a markedly
attenuated sensitivity to behavioral consequences of stress, in that
they were insensitive to the normal anxiogenic-like effect of restraint
stress on the elevated plus maze and displayed absent fear suppression
of behavior in a punished drinking test. (
ii
) A
selective impairment of spatial memory acquisition was found in the
Morris water maze. Control experiments suggest these traits to be
independent. These phenotypic traits were accompanied by an
overexpression of prepro-NPY mRNA and NPY peptide and decreased NPY-Y1
binding within the hippocampus, a brain structure implicated both in
memory processing and stress responses. Data obtained using this unique
model support and extend a previously postulated anti-stress action of
NPY and provide novel evidence for a role of NPY in learning and
memory.
Purification, molecular cloning, and sequence analysis of sucrose-6
F
-phosphate phosphohydrolase from plants
Authors: Lunn John E. , Ashton Anthony R. , Hatch Marshall D. , Heldt Hans W.
Published: July 2002

Sucrose-6
F
-phosphate phosphohydrolase (SPP; EC
3.1.3.24
) catalyzes the final step in the pathway of sucrose
biosynthesis and is the only enzyme of photosynthetic carbon
assimilation for which the gene has not been identified. The enzyme was
purified to homogeneity from rice (
Oryza sativa
L.)
leaves and partially sequenced. The rice leaf enzyme is a dimer with a
native molecular mass of 100 kDa and a subunit molecular mass of 50
kDa. The enzyme is highly specific for sucrose 6
F
-phosphate
with a
K
m
of 65 μM and a specific activity
of 1250 μmol min
−1
mg
−1
protein. The
activity is dependent on Mg
2+
with a remarkably low
K
a
of 8–9 μM and is weakly inhibited by
sucrose. Three peptides from cleavage of the purified rice SPP with
endoproteinase Lys-C showed similarity to the deduced amino acid
sequences of three predicted open reading frames (ORF) in the
Arabidopsis thaliana
genome and one in the genome of the
cyanobacterium
Synechocystis
sp. PCC6803, as well as
cDNA clones from
Arabidopsis
, maize, and other species
in the GenBank database of expressed sequence tags. The putative maize
SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide.
Heterologous expression in
Escherichia coli
showed that
this cDNA clone encoded a functional SPP enzyme. The 260-amino acid
N-terminal catalytic domain of the maize SPP is homologous to the
C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of
the GenBank database indicated that the maize SPP is a member of the
haloacid dehalogenase hydrolase/phosphatase superfamily.
HIV-1 protease cleaves eukaryotic initiation factor 4G and inhibits cap-dependent translation
Authors: Ventoso Iván , Blanco Raquel , Perales Celia , Carrasco Luis
Published: July 2002

Several animal viruses inhibit host protein synthesis, but only some members of the picornavirus group are known to do so by cleaving translation initiation factor eIF4G. Here we report that infection of human CD4
+
cells with HIV-1 also leads to proteolysis of eIF4G and profound inhibition of cellular translation. Purified HIV-1 protease directly cleaves eIF4GI at positions 678, 681, and 1086, separating the three domains of this initiation factor. Proteolysis of eIF4GI by HIV-1 protease, as with poliovirus 2A protease, inhibits protein synthesis directed by capped mRNAs but allows internal ribosome entry site-driven translation. These findings indicate that HIV-1, a member of retrovirus group, shares with picornaviruses the capacity to proteolyze eIF4G.
UniGene cDNA array-based monitoring of transcriptome changes during mouse placental development
Authors: Hemberger Myriam , Cross James C. , Ropers Hans-Hilger , Lehrach Hans , Fundele Reinald , Himmelbauer Heinz
Published: July 2002

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and
in situ
hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive
in situ
hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.
Cross-talk between sympathetic neurons and adipocytes in coculture
Authors: Turtzo L. Christine , Marx Ruth , Lane M. Daniel
Published: July 2002

White adipose tissue plays an integral role in energy metabolism and is governed by endocrine, autocrine, and neural signals. Neural control of adipose metabolism is mediated by sympathetic neurons that innervate the tissue. To investigate the effects of this innervation, an
ex vivo
system was developed in which 3T3-L1 adipocytes are cocultured with sympathetic neurons isolated from the superior cervical ganglia of newborn rats. In coculture, both adipocytes and neurons exhibit appropriate morphology, express cell-type-specific markers, and modulate key metabolic processes in one another. Lipolysis (stimulated by β-adrenergic agents) and leptin secretion by adipocytes are down-regulated by neurons in coculture, effects apparently mediated by neuropeptide Y (NPY). Secretion of NPY by neurons is up-regulated dramatically by the presence of adipocytes in coculture and appears to be mediated by an adipocyte-derived soluble factor. Insulin, an antilipolytic agent, down-regulates NPY secretion. Our findings suggest that an adipocyte-derived factor(s) up-regulates the secretion of NPY by sympathetic neurons, which, in turn, attenuates lipolytic energy mobilization by adipocytes.
Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of
P
-glycoprotein
Authors: Kondratov Roman V. , Komarov Pavel G. , Becker Yigal , Ewenson Ariel , Gudkov Andrei V.
Published: July 2002

By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing
P
-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.
Transformations in methane hydrates
Authors: Chou I-Ming , Sharma Anurag , Burruss Robert C. , Shu Jinfu , Mao Ho-kwang , Hemley Russell J. , Goncharov Alexander F. , Stern Laura A. , Kirby Stephen H.
Published: July 2002

Detailed study of pure methane hydrate in a diamond cell within situoptical, Raman, and x-ray microprobe techniques reveals two previously unknown structures, structure II and structure H, at high pressures. The structure II methane hydrate at 250 MPa has a cubic unit cell ofa= 17.158(2) Å and volumeV= 5051.3(13) Å3; structure H at 600 MPa has a hexagonal unit cell ofa= 11.980(2) Å,c= 9.992(3) Å, andV= 1241.9(5) Å3. The compositions of these two investigated phases are still not known. With the effects of pressure and the presence of other gases in the structure, the structure II phase is likely to dominate over the known structure I methane hydrate within deep hydrate-bearing sediments underlying continental margins.
betawrap
: Successful prediction of parallel β-helices from primary sequence reveals an association with many microbial pathogens
Authors: Bradley Phil , Cowen Lenore , Menke Matthew , King Jonathan , Berger Bonnie
Published: July 2002

The amino acid sequence rules that specify β-sheet structure in
proteins remain obscure. A subclass of β-sheet proteins,
parallel β-helices, represent a processive folding of the chain into
an elongated topologically simpler fold than globular β-sheets. In
this paper, we present a computational approach that predicts the
right-handed parallel β-helix supersecondary structural motif in
primary amino acid sequences by using β-strand interactions learned
from non-β-helix structures. A program called
BETAWRAP
(
http://theory.lcs.mit.edu/betawrap
) implements this
method and recognizes each of the seven known parallel β-helix
families, when trained on the known parallel β-helices from outside
that family. BETAWRAP identifies 2,448 sequences
among 595,890 screened from the National Center for Biotechnology
Information (NCBI;
http://www.ncbi.nlm.nih.gov/
) nonredundant
protein database as likely parallel β-helices. It identifies
surprisingly many bacterial and fungal protein sequences that play a
role in human infectious disease; these include toxins, virulence
factors, adhesins, and surface proteins of
Chlamydia
,
Helicobacteria
,
Bordetella
,
Leishmania
,
Borrelia
,
Rickettsia
,
Neisseria
, and
Bacillus anthracis
. Also unexpected was the rarity of
the parallel β-helix fold and its predicted sequences among higher
eukaryotes. The computational method introduced here can be called a
three-dimensional dynamic profile method because it generates
interstrand pairwise correlations from a processive sequence wrap. Such
methods may be applicable to recognizing other beta structures for
which strand topology and profiles of residue accessibility are well
conserved.
An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts.
Authors: Lee T H , Kirschner M W
Published: July 2002

The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition.
Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.
Authors: Makarov S S , Olsen J C , Johnston W N , Anderle S K , Brown R R , Baldwin A S , Haskill J S , Schwab J H
Published: July 2002

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.
Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.
Authors: Larionov V , Kouprina N , Graves J , Chen X N , Korenberg J R , Resnick M A
Published: July 2002

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.
A mutation that allows endosperm development without fertilization.
Authors: Ohad N , Margossian L , Hsu Y C , Williams C , Repetti P , Fischer R L
Published: July 2002

The mechanisms that initiate reproductive development after fertilization are not understood. Reproduction in higher plants is unique because it is initiated by two fertilization events in the haploid female gametophyte. One sperm nucleus fertilizes the egg to form the embryo. A second sperm nucleus fertilizes the central cell to form the endosperm, a unique tissue that supports the growth of the embryo. Fertilization also activates maternal tissue differentiation, the ovule integuments form the seed coat, and the ovary forms the fruit. To investigate mechanisms that initiate reproductive development, a female-gametophytic mutation termed fie (fertilization-independent endosperm) has been isolated in Arabidopsis. The fie mutation specifically affects the central cell, allowing for replication of the central cell nucleus and endosperm development without fertilization. The fie mutation does not appear to affect the egg cell, suggesting that the processes that control the initiation of embryogenesis and endosperm development are different. FIE/fie seed coat and fruit undergo fertilization-independent differentiation, which shows that the fie female gametophyte is the source of signals that activates sporophytic fruit and seed coat development. The mutant fie allele is not transmitted by the female gametophyte. Inheritance of the mutant fie allele by the female gametophyte results in embryo abortion, even when the pollen bears the wild-type FIE allele. Thus, FIE carries out a novel, essential function for female reproductive development.
Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.
Authors: Mason H S , Ball J M , Shi J J , Jiang X , Estes M K , Arntzen C J
Published: July 2002

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.
The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.
Authors: Junghans R P , Anderson C L
Published: July 2002

More than 30 years ago, Brambell published the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, 1. C. (1964) Nature (London) 203, 1352-1355] that remains as the cornerstone for thinking on IgG catabolism. To explain the long survival of IgG relative to other plasma proteins and its pattern of increased fractional catabolism with high concentrations of IgG, Brambell postulated specific IgG "protection receptors" (FcRp) that would bind IgG in pinocytic vacuoles and redirect its transport to the circulation; when the FcRp was saturated, the excess unbound IgG then would pass to unrestricted lysosomal catabolism. Brambell subsequently postulated the neonatal gut transport receptor (FcRn) and showed its similar saturable character. FcRn was recently cloned but FcRp has not been identified. Using a genetic knockout that disrupts the FcRn and intestinal IgG transport, we show that this lesion also disrupts the IgG protection receptor, supporting the identity of these two receptors. IgG catabolism was 10-fold faster and IgG levels were correspondingly lower in mutant than in wild-type mice, whereas IgA was the same between groups, demonstrating the specific effects on the IgG system. Disruption of the FcRp in the mutant mice was also shown to abrogate the classical pattern of decreased IgG survival with higher IgC concentration. Finally, studies in normal mice with monomeric antigen-antibody complexes showed differential catabolism in which antigen dissociates in the endosome and passes to the lysosome, whereas the associated antibody is returned to circulation; in mutant mice, differential catabolism was lost and the whole complex cleared at the same accelerated rate as albumin, showing the central role of the FcRp to the differential catabolism mechanism. Thus, the same receptor protein that mediates the function of the FcRn transiently in the neonate is shown to have its functionally dominant expression as the FcRp throughout life, resolving a longstanding mystery of the identity of the receptor for the protection of IgG. This result also identifies an important new member of the class of recycling surface receptors and enables the design of protein adaptations to exploit this mechanism to improve survivals of other therapeutic proteins in vivo.