bluuebunny/crossref_metadata_2025 huggingface dataset:
Point mutations in a nucleoside transporter gene from
Leishmania donovani
confer drug resistance and alter substrate selectivity
Authors: Vasudevan Gayatri , Ullman Buddy , Landfear Scott M.
Published: July 2002

Leishmania
parasites lack a purine biosynthetic
pathway and depend on surface nucleoside and nucleobase transporters to
provide them with host purines.
Leishmania donovani
possess two closely related genes that encode high affinity
adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and
that transport the toxic adenosine analog tubercidin in addition to the
natural substrates. In this study, we have characterized a
drug-resistant clonal mutant of
L. donovani
(TUBA5) that
is deficient in LdNT1 transport and consequently resistant to
tubercidin. In TUBA5 cells, the
LdNT1.2
genes had the
same sequence as wild-type cells. However, because
LdNT1.2
mRNA is not detectable in either wild-type or
TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside
transport in this stage of the life cycle. In contrast, the TUBA5 cells
were compound heterozygotes at the
LdNT1.1
locus
containing two mutant alleles that encompassed distinct point
mutations, each of which impaired transport function. One of the mutant
LdNT1.1
alleles encoded a G183D substitution in
predicted TM 5, and the other allele contained a C337Y change in
predicted TM 7. Whereas G183D and C337Y mutants had only slightly
elevated adenosine
K
m
values, the severe
impairment in transport resulted from drastically (≈20-fold) reduced
V
max
values. Because these transporters were
correctly targeted to the plasma membrane, the reduction in
V
max
apparently resulted from a defect in
translocation. Strikingly, G183 was essential for pyrimidine nucleoside
but not adenosine transport. A mutant transporter with a G183A
substitution had an altered substrate specificity, exhibiting robust
adenosine transport but undetectable uridine uptake. These results
suggest that TM 5 is likely to form part of the nucleoside
translocation pathway in LdNT1.1
A dynamic programming algorithm for haplotype block partitioning
Authors: Zhang Kui , Deng Minghua , Chen Ting , Waterman Michael S. , Sun Fengzhu
Published: July 2002

We develop a dynamic programming algorithm for haplotype block partitioning to minimize the number of representative single nucleotide polymorphisms (SNPs) required to account for most of the common haplotypes in each block. Any measure of haplotype quality can be used in the algorithm and of course the measure should depend on the specific application. The dynamic programming algorithm is applied to analyze the chromosome 21 haplotype data of Patil
et al.
[Patil, N., Berno, A. J., Hinds, D. A., Barrett, W. A., Doshi, J. M., Hacker, C. R., Kautzer, C. R., Lee, D. H., Marjoribanks, C., McDonough, D. P.,
et al.
(2001)
Science
294, 1719–1723], who searched for blocks of limited haplotype diversity. Using the same criteria as in Patil
et al.
, we identify a total of 3,582 representative SNPs and 2,575 blocks that are 21.5% and 37.7% smaller, respectively, than those identified using a greedy algorithm of Patil
et al
. We also apply the dynamic programming algorithm to the same data set based on haplotype diversity. A total of 3,982 representative SNPs and 1,884 blocks are identified to account for 95% of the haplotype diversity in each block.
Prothrombotic phenotype of protein Z deficiency
Authors: Yin Zheng-Feng , Huang Zhong-Fu , Cui Jisong , Fiehler Ryan , Lasky Nina , Ginsburg David , Broze George J.
Published: July 2002

Protein Z (PZ) is a vitamin K-dependent plasma protein whose function has been uncertain. The structure of PZ is very similar to that of the coagulation-related factors VII, IX, and X and PC, but PZ differs from these other proteins in that it is not the zymogen of a serine protease. We have shown recently that PZ forms a calcium ion-dependent complex with activated factor X at phospholipid surfaces and that this interaction leads to the inhibition of activated factor X activity through, in part, the action of a previously unidentified plasma protein named PZ-dependent protease inhibitor. Herein, we report that the presence of PZ dampens the coagulation response in human plasma and that concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor V
Leiden
mice. The results indicate that PZ plays a physiologically important role in the regulation of coagulation.
The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility
Authors: Shestakova Elena A. , Singer Robert H. , Condeelis John
Published: July 2002

β-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the β-actin mRNA near the leading edge is facilitated by a short sequence in the 3′ untranslated region, the “zip code.” Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3′ untranslated region leads to delocalization of β-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after β-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of β-actin protein. These results indicate that delocalization of β-actin mRNA results in delocalization of nucleation sites and β-actin protein from the leading edge followed by loss of cell polarity and directional movement.
Direct, activating interaction between glycogen synthase kinase-3β and p53 after DNA damage
Authors: Watcharasit Piyajit , Bijur Gautam N. , Zmijewski Jaroslaw W. , Song Ling , Zmijewska Anna , Chen Xinbin , Johnson Gail V. W. , Jope Richard S.
Published: July 2002

Glycogen synthase kinase-3β (GSK3β) is a central figure in Wnt signaling, in which its activity is controlled by regulatory binding proteins. Here we show that binding proteins outside the Wnt pathway also control the activity of GSK3β. DNA damage induced by camptothecin, which activates the tumor suppressor p53, was found to activate GSK3β. This activation occurred by a phosphorylation-independent mechanism involving direct binding of GSK3β to p53, which was confined to the nucleus where p53 is localized, and mutated p53 (R175H) bound but did not activate GSK3β. Activation of GSK3 promoted responses to p53 including increases in p21 levels and caspase-3 activity. Thus, after DNA damage there is a direct interaction between p53 and GSK3β, and these proteins act in concert to regulate cellular responses to DNA damage.
A novel application of gene arrays:
Escherichia coli
array provides insight into the biology of the obligate endosymbiont of tsetse flies
Authors: Akman Leyla , Aksoy Serap
Published: July 2002

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms
in vitro
. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont,
Wigglesworthia glossinidia,
a member of the Enterobacteriaceae
,
closely related to
Escherichia coli
, for fertility and possibly nutrition. We show here that the intracellular
Wigglesworthia
has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for
E. coli
. We were able to identify 650 orthologous genes in
Wigglesworthia
corresponding to ≈85% of its genome. The arrays were also applied for expression analysis using
Wigglesworthia
cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of
Wigglesworthia
. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that
Wigglesworthia
may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of
E. coli
gene arrays to obtain broad genome information for a closely related organism in the absence of complete genome sequence data.
Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant
p53
transcripts
Authors: Watanabe Takashi , Sullenger Bruce A.
Published: July 2002

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt)
p53
cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the
p53
gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with
trans
-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective
p53
mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (
MDR
1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in
MDR
1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.
Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion
Authors: González-Reyes Luis , Ruiz-Argüello M. Begoña , García-Barreno Blanca , Calder Leslie , López Juan A. , Albar Juan P. , Skehel John J. , Wiley Don C. , Melero José A.
Published: July 2002

Preparations of purified full-length fusion (F) protein of human
respiratory syncytial virus (HRSV) expressed in recombinant vaccinia-F
infected cells, or of an anchorless mutant (F

TM
−

)
lacking the C-terminal 50 amino acids secreted from
vaccinia-F

TM
−

-infected cells contain a minor
polypeptide that is an intermediate product of proteolytic processing
of the F protein precursor F0. N-terminal sequencing of the
intermediate demonstrated that it is generated by cleavage at a
furin-motif, residues 106–109 of the F sequence. By contrast, the F1 N
terminus derives from cleavage at residue 137 of F0 which is also
C-terminal to a furin recognition site at residues 131–136.
Site-directed mutagenesis indicates that processing of F0 protein
involves independent cleavage at both sites. Both cleavages are
required for the F protein to be active in membrane fusion as judged by
syncytia formation, and they allow changes in F structure from cone- to
lollipop-shaped spikes and the formation of rosettes by anchorless F.
Peripheral anti-Aβ antibody alters CNS and plasma Aβ clearance and decreases brain Aβ burden in a mouse model of Alzheimer's disease
Authors: DeMattos Ronald B. , Bales Kelly R. , Cummins David J. , Dodart Jean-Cosme , Paul Steven M. , Holtzman David M.
Published: July 2002

Active immunization with the amyloid β (Aβ) peptide has been shown to decrease brain Aβ deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-Aβ antibodies were shown to mimic this effect. In exploring factors that alter Aβ metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of Aβ was able to bind and completely sequester plasma Aβ. Peripheral administration of m266 to PDAPP transgenic mice, in which Aβ is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma Aβ, due, in part, to a change in Aβ equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces Aβ deposition, m266 did not bind to Aβ deposits in the brain. Thus, m266 appears to reduce brain Aβ burden by altering CNS and plasma Aβ clearance.
Bootstrapping cluster analysis: Assessing the reliability of conclusions from microarray experiments
Authors: Kerr M. Kathleen , Churchill Gary A.
Published: July 2002

We introduce a general technique for making statistical inference from clustering tools applied to gene expression microarray data. The approach utilizes an analysis of variance model to achieve normalization and estimate differential expression of genes across multiple conditions. Statistical inference is based on the application of a randomization technique, bootstrapping. Bootstrapping has previously been used to obtain confidence intervals for estimates of differential expression for individual genes. Here we apply bootstrapping to assess the stability of results from a cluster analysis. We illustrate the technique with a publicly available data set and draw conclusions about the reliability of clustering results in light of variation in the data. The bootstrapping procedure relies on experimental replication. We discuss the implications of replication and good design in microarray experiments.
Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1receptor
Authors: Johansson Björn , Halldner Linda , Dunwiddie Thomas V. , Masino Susan A. , Poelchen Wolfgang , Giménez-Llort Lydia , Escorihuela Rosa M. , Fernández-Teruel Alberto , Wiesenfeld-Hallin Zsuzsanna , Xu Xiao-Jun , Hårdemark Anna , Betsholtz Christer , Herlenius Eric , Fredholm Bertil B.
Published: July 2002

Caffeine is believed to act by blocking adenosine A1and A2Areceptors (A1R, A2AR), indicating that some A1receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A1R (A1R−/−). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A1R+/+mice, but A1R−/−mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A1R−/−mice. In A1R+/−mice the potency of adenosine was halved, as was the number of A1R. In A1R−/−mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A1Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.
Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27
KIP1
Authors: Henriet Patrick , Zhong Zhi-Duan , Brooks Peter C. , Weinberg Kenneth I. , DeClerck Yves A.
Published: July 2002

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G
1
/S checkpoint and maintain high levels of p27
KIP1
mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27
KIP1
is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α
2
β
1
, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.
Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor
Authors: Dumoutier Laure , Van Roost Emiel , Colau Didier , Renauld Jean-Christophe
Published: July 2002

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory responsein vivo.
Immune hyporesponsiveness to amyloid β-peptide in amyloid precursor protein transgenic mice: Implications for the pathogenesis and treatment of Alzheimer's disease
Authors: Monsonego Alon , Maron Ruth , Zota Victor , Selkoe Dennis J. , Weiner Howard L.
Published: July 2002

Alzheimer's disease is a dementia that involves progressive deposition of amyloid β-protein (Aβ) in brain regions important for memory and cognition, followed by secondary inflammation that contributes to the neuropathologic process. Immunization with Aβ can reduce cerebral Aβ burden and consequent neuropathologic changes in the brains of mice transgenic for the β-amyloid precursor protein (APP). We found that transgenic expression of human APP in B6SJL mice, under the prion promoter, results in immune hyporesponsiveness to human Aβ, in terms of both antibody and cellular immune responses. The decreased antibody responses were related not to B cell tolerance but rather to the inability of Aβ-specific T cells to provide help for antibody production. The immune hyporesponsiveness could be overcome if T cell help was provided by coupling an Aβ B cell epitope to BSA. Our results suggest that expression of APP in transgenic mice is associated with an Aβ-specific impaired adaptive immune response that may contribute to the neuropathology. Moreover, humans with life-long elevation of brain and peripheral Aβ (e.g., patients with presenilin mutations or Down syndrome) could have reduced immune responses to Aβ vaccination.
The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species
Authors: Ruefli Astrid A. , Ausserlechner Michael J. , Bernhard David , Sutton Vivien R. , Tainton Kellie M. , Kofler Reinhard , Smyth Mark J. , Johnstone Ricky W.
Published: July 2002

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome
c
release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.
Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis
Authors: Nakamura Hajime , Herzenberg Leonore A. , Bai Jie , Araya Shinichi , Kondo Norihiko , Nishinaka Yumiko , Herzenberg Leonard A. , Yodoi Junji
Published: July 2002

Thioredoxin (Trx), a redox enzyme with a conserved active site (Cys-32–Gly–Pro–Cys-35), is induced and secreted into circulation in response to inflammation. Studies here demonstrate that elevating Trx levels in circulation either by i.v. injection of recombinant Trx or stimulating Trx release in Trx-transgenic mice dramatically blocks lipopolysaccharide (LPS)-stimulated neutrophil migration in the murine air pouch chemotaxis model. Furthermore, we show that leukocyte recruitment induced by the murine chemokines KC/GROα, RANTES (regulated upon activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) is suppressed also in Trx-transgenic mice. Addressing the mechanism responsible for this suppression, we show that circulating Trx blocks (i) the LPS-stimulatedin vitroactivation of neutrophil p38 mitogen-activated protein kinase, (ii) the normal down-regulation of CD62L on neutrophils migrating into the LPS-stimulated air pouch, and (iii) thein vitroadhesion of LPS-activated neutrophils on endothelial cells. However, as we also show, Trx does not alter the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD62P, and CD62E) within 3 h. Collectively, these findings indicate that elevated levels of circulating Trx interfere with chemotaxis by acting directly on neutrophils. We discuss these findings in the context of recent studies reporting beneficial effects of acutely elevated Trx in ischemic injury and negative effects associated with chronically elevated Trx in HIV disease.
γ-Tocopherol and its major metabolite, in contrast to α-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells
Authors: Jiang Qing , Elson-Schwab Ilan , Courtemanche Chantal , Ames Bruce N.
Published: July 2002

Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin
E
2
(PGE
2
) plays a key role in inflammation
and its associated diseases, such as cancer and vascular heart disease.
Here we report that γ-tocopherol (γT) reduced PGE
2
synthesis in both lipopolysaccharide (LPS)-stimulated RAW264.7
macrophages and IL-1β-treated A549 human epithelial cells with an
apparent IC
50
of 7.5 and 4 μM, respectively. The major
metabolite of dietary γT,
2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC), also
exhibited an inhibitory effect, with an IC
50
of ≈30 μM
in these cells. In contrast, α-tocopherol at 50 μM slightly reduced
(25%) PGE
2
formation in macrophages, but had no effect in
epithelial cells. The inhibitory effects of γT and γ-CEHC stemmed
from their inhibition of COX-2 activity, rather than affecting protein
expression or substrate availability, and appeared to be independent of
antioxidant activity. γ-CEHC also inhibited PGE
2
synthesis when exposed for 1 h to COX-2-preinduced cells followed
by the addition of arachidonic acid (AA), whereas under similar
conditions, γT required an 8- to 24-h incubation period to cause the
inhibition. The inhibitory potency of γT and γ-CEHC was diminished
by an increase in AA concentration, suggesting that they might compete
with AA at the active site of COX-2. We also observed a moderate
reduction of nitrite accumulation and suppression of inducible nitric
oxide synthase expression by γT in lipopolysaccharide-treated
macrophages. These findings indicate that γT and its major metabolite
possess anti-inflammatory activity and that γT at physiological
concentrations may be important in human disease prevention.
Contribution of individual random mutations to genotype-by-environment interactions in
Escherichia coli
Authors: Remold Susanna K. , Lenski Richard E.
Published: July 2002

Numerous studies have shown genotype-by-environment (G×E)
interactions for traits related to organismal fitness. However, the
genetic architecture of the interaction is usually unknown because
these studies used genotypes that differ from one another by many
unknown mutations. These mutations were also present as standing
variation in populations and hence had been subject to prior selection.
Based on such studies, it is therefore impossible to say what fraction
of new, random mutations contributes to G×E interactions. In this
study, we measured the fitness in four environments of 26 genotypes of
Escherichia coli
, each containing a single random
insertion mutation. Fitness was measured relative to their common
progenitor, which had evolved on glucose at 37°C for the preceding
10,000 generations. The four assay environments differed in limiting
resource and temperature (glucose, 28°C; maltose, 28°C; glucose,
37°C; and maltose, 37°C). A highly significant interaction between
mutation and resource was found. In contrast, there was no interaction
involving temperature. The resource interaction reflected much higher
among mutation variation for fitness in maltose than in glucose. At
least 11 mutations (42%) contributed to this G×E interaction through
their differential fitness effects across resources. Beneficial
mutations are generally thought to be rare but, surprisingly, at least
three mutations (12%) significantly improved fitness in maltose, a
resource novel to the progenitor. More generally, our findings
demonstrate that G×E interactions can be quite common, even for
genotypes that differ by only one mutation and in environments
differing by only a single factor.
Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors
Authors: Medema J. P. , de Jong J. , Peltenburg L. T. C. , Verdegaal E. M. E. , Gorter A. , Bres S. A. , Franken K. L. M. C. , Hahne M. , Albar J. P. , Melief C. J. M. , Offringa R.
Published: July 2002

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing bothin vitroandin vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.
Receptor-mediated endocytosis of soluble and membrane-tethered Sonic hedgehog by Patched-1
Authors: Incardona John P. , Lee Ju Ho , Robertson Christie P. , Enga Kristi , Kapur Raj P. , Roelink Henk
Published: July 2002

Patched (Ptc) is the ligand-binding component of the Hedgehog (Hh)
receptor complex. In the
Drosophila
embryo, Ptc and Hh
colocalize in vesicular punctate structures. However, receptor-mediated
endocytosis of Hh proteins has not been demonstrated. By using chick
neural plate explants, we show that Sonic hedgehog (Shh)-responsive
neural precursor cells internalize recombinant and
endogenous Shh and provide direct evidence for a gradient
of endogenous Shh in the ventral neural tube. Shh
internalization is blocked by a monoclonal antibody whose epitope
overlaps the Ptc-binding site of Shh. These findings suggest that Shh
internalization is mediated by Ptc-1 and may be linked to signaling.
Concordantly, transfection of mammalian cell lines with a Ptc-1 cDNA
confers the ability to internalize multiple forms of Shh, including
transmembrane-anchored Shh, by a dynamin-dependent process.
SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infectionin vitroandin vivo
Authors: Strizki Julie M. , Xu Serena , Wagner Nicole E. , Wojcik Lisa , Liu Jia , Hou Yan , Endres Michael , Palani Anandan , Shapiro Sherry , Clader John W. , Greenlee William J. , Tagat Jayaram R. , McCombie Stuart , Cox Kathleen , Fawzi Ahmad B. , Chou Chuan-Chu , Pugliese-Sivo Catherine , Davies Liza , Moreno Mary E. , Ho David D. , Trkola Alexandra , Stoddart Cheryl A. , Moore John P. , Reyes Gregory R. , Baroudy Bahige M.
Published: July 2002

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime–piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activityin vitroagainst primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50–60% and a serum half-life of 5–6 h. On the basis of its novel mechanism of action, potent antiviral activity, andin vivopharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.
Transgenic mice produced by retroviral transduction of male germ-line stem cells
Authors: Nagano Makoto , Brinster Clayton J. , Orwig Kyle E. , Ryu Buom-Yong , Avarbock Mary R. , Brinster Ralph L.
Published: July 2002

Male germ-line stem cells are the only cell type in postnatal mammals that have the capability to self-renew and to contribute genes to the next generation. Genetic modification of these cells would provide an opportunity to study the biology of their complex self-renewal and differentiation processes, as well as enable the generation of transgenic animals in a wide range of species. Although retroviral vectors have been used as an efficient method to introduce genes into a variety of cell types, postnatal male germ-line stem cells have seemed refractory to direct infection by these viruses. In addition, expression of genes transduced into several types of stem cells, such as embryonic or hematopoietic, is often attenuated or silenced. We demonstrate here thatin vitroretroviral-mediated gene delivery into spermatogonial stem cells of both adult and immature mice results in stable integration and expression of a transgene in 2–20% of stem cells. After transplantation of the transduced stem cells into the testes of infertile recipient mice, approximately 4.5% of progeny from these males are transgenic, and the transgene is transmitted to and expressed in subsequent generations. Therefore, there is no intrinsic barrier to retroviral transduction in this stem cell, and transgene expression is not extinguished after transmission to progeny.
Stereotyped and specific gene expression programs in human innate immune responses to bacteria
Authors: Boldrick Jennifer C. , Alizadeh Ash A. , Diehn Maximilian , Dudoit Sandrine , Liu Chih Long , Belcher Christopher E. , Botstein David , Staudt Louis M. , Brown Patrick O. , Relman David A.
Published: July 2002

The innate immune response is crucial for defense against microbial pathogens. To investigate the molecular choreography of this response, we carried out a systematic examination of the gene expression program in human peripheral blood mononuclear cells responding to bacteria and bacterial products. We found a remarkably stereotyped program of gene expression induced by bacterial lipopolysaccharide and diverse killed bacteria. An intricately choreographed expression program devoted to communication between cells was a prominent feature of the response. Other features suggested a molecular program for commitment of antigen-presenting cells to antigens captured in the context of bacterial infection. Despite the striking similarities, there were qualitative and quantitative differences in the responses to different bacteria. Modulation of this host-response program by bacterial virulence mechanisms was an important source of variation in the response to different bacteria.
Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters
Authors: Iyer M. , Wu L. , Carey M. , Wang Y. , Smallwood A. , Gambhir S. S.
Published: July 2002

We are developing assays to image tissue-specific reporter gene
expression in living mice by using optical methods and positron
emission tomography. Approaches for imaging reporter gene
expression depend on robust levels of mRNA and reporter protein.
Attempts to image reporter gene expression driven by weak promoters are
often hampered by the poor transcriptional activity of such promoters.
Most tissue-specific promoters are weak relative to stronger but
constitutively expressing viral promoters. In this study, we have
validated methods to enhance the transcriptional activity of the
prostate-specific antigen promoter for imaging by using a two-step
transcriptional amplification (TSTA) system. We used the TSTA system to
amplify expression of firefly luciferase (
fl
) and mutant
herpes simplex virus type 1 thymidine kinase
(HSV1-sr39
tk
) in a prostate cancer cell line (LNCaP). We
demonstrate ≈50-fold (
fl
) and ≈12-fold
(HSV1-sr39
tk
) enhancement by using the two-step
approach. The TSTA system is observed to retain tissue selectivity. A
cooled charge-coupled device optical imaging system was used to
visualize the amplified
fl
expression in living mice
implanted with LNCaP cells transfected
ex vivo
. These
imaging experiments reveal a ≈5-fold gain in imaging signal by using
the TSTA system over the one-step system. The TSTA approach will be a
valuable and generalizable tool to amplify and noninvasively image
reporter gene expression in living animals by using tissue-specific
promoters. The approaches validated should have important implications
for study of gene therapy vectors, cell trafficking, transgenic models,
as well as studying development of eukaryotic organisms.
Structural organization of yeast and mammalian mediator complexes
Authors: Dotson Michaela R. , Yuan Chao Xing , Roeder Robert G. , Myers Lawrence C. , Gustafsson Claes M. , Jiang Yi Wei , Li Yang , Kornberg Roger D. , Asturias Francisco J.
Published: July 2002

Structures of yeast Mediator complex, of a related complex from
mouse cells and of thyroid hormone receptor-associated protein complex
from human cells have been determined by three-dimensional
reconstruction from electron micrographs of single particles. All three
complexes show a division in two parts, a “head” domain and a
combined “middle-tail” domain. The head domains of the three
complexes appear most similar and interact most closely with RNA
polymerase II. The middle-tail domains show the greatest structural
divergence and, in the case of the tail domain, may not interact with
polymerase at all. Consistent with this structural divergence, analysis
of a yeast Mediator mutant localizes subunits that are not conserved
between yeast and mammalian cells to the tail domain. Biochemically
defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to
the middle and head domains.
Identification of ligands and coligands for the ecdysone-regulated gene switch
Authors: Saez Enrique , Nelson Michael C. , Eshelman Brian , Banayo Ester , Koder Alan , Cho Gerald J. , Evans Ronald M.
Published: July 2002

The ecdysone-inducible gene switch is a useful tool for modulating
gene expression in mammalian cells and transgenic animals. We have
identified inducers derived from plants as well as certain classes of
insecticides that increase the versatility of this gene regulation
system. Phytoecdysteroids share the favorable kinetics of steroids, but
are inert in mammals. The gene regulation properties of one of these
ecdysteroids have been examined in cell culture and in newly developed
strains of ecdysone-system transgenic mice. Ponasterone A is a potent
regulator of gene expression in cells and transgenic animals, enabling
reporter genes to be turned on and off rapidly. A number of
nonsteroidal insecticides have been identified that also activate the
ecdysone system. Because the gene-controlling properties of the
ecdysone switch are based on a heterodimer composed of a modified
ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have
tested the effect of RXR ligands on the VgEcR/RXR complex. Used
alone, RXR ligands display no activity on the ecdysone switch. However,
when used in combination with a VgEcR ligand, RXR ligands dramatically
enhance the absolute levels of induction. This property of the
heterodimer has allowed the development of superinducer combinations
that increase the dynamic range of the system.
Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.
Authors: Sitnikov D M , Schineller J B , Baldwin T O
Published: July 2002

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.
Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.
Authors: Belcourt M F , Hodnick W F , Rockwell S , Sartorelli A C
Published: July 2002

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.
The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.
Authors: Mourelatos Z , Gonatas N K , Stieber A , Gurney M E , Dal Canto M C
Published: July 2002

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.
Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.
Authors: Kuspa A , Loomis W F
Published: July 2002

High resolution gene maps of the six chromosomes of Dictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34-magabase pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes and should facilitate more detailed analyses of this genome. cost of silicon-based construction and in the efficient sample handling afforded by component integration.
A tyrosine kinase profile of prostate carcinoma.
Authors: Robinson D , He F , Pretlow T , Kung H J
Published: July 2002

Tyrosine kinases play central roles in the growth and differentiation of normal and tumor cells. In this study, we have analyzed the general tyrosine kinase expression profile of a prostate carcinoma (PCA) xenograft, CWR22. We describe here an improved reverse transcriptase-PCR approach that permits identification of nearly 40 different kinases in a single screening; several of these kinases are newly cloned kinases and some are novel. According to this, there are 11 receptor kinases, 9 nonreceptor kinases, and at least 7 dual kinases expressed in the xenograft tissue. The receptor kinases include erbB2, erbB3, Ret, platelet-derived growth factor receptor, sky, nyk, eph, htk, sek (eph), ddr, and tkt. The nonreceptor kinases are lck, yes, abl, arg, JakI, tyk2, and etk/bmx. Most of the dual kinases are in the mitogen-activating protein (MAP) kinase-kinase (MKK) family, which includes MKK3, MKK4, MEK5, and a novel one. As a complementary approach, we also analyzed by specific reverse transcriptase-PCR primers the expression profile of erbB/epidermal growth factor receptor family receptors in a variety of PCA specimens, cell lines, and benign prostatic hyperplasia. We found that erbB1, -2, and -3 are often coexpressed in prostate tissues, but not in erbB4. The information established here should provide a base line to study the possible growth and oncogenic signals of PCA.