bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiangiogenic gene therapy targeting the endothelium-specific receptor tyrosine kinase Tie2
Authors: Lin Pengnian , Buxton Jake A. , Acheson Ann , Radziejewski Czeslaw , Maisonpierre Peter C. , Yancopoulos George D. , Channon Keith M. , Hale Laura P. , Dewhirst Mark W. , George Samuel E. , Peters Kevin G.
Published: July 2002

Angiogenesis is required for tumor growth and metastasis, and inhibition of angiogenesis is a promising approach for anticancer therapy. Tie2 (a.k.a Tek) is an endothelium-specific receptor tyrosine kinase known to play a role in tumor angiogenesis. To explore the therapeutic potential of blocking the Tie2 pathway, an adenoviral vector was constructed to deliver a recombinant, soluble Tie2 receptor (AdExTek) capable of blocking Tie2 activation. Two days after i.v. injection of AdExTek, the plasma concentration of ExTek exceeded 1 mg/ml and was maintained for about 8 days. Administration of AdExTek to mice with two different well established primary tumors, a murine mammary carcinoma (4T1) or a murine melanoma (B16F10.9), significantly inhibited the growth rate of both tumors (64% and 47%, respectively). To study the effect of ExTek on tumor metastasis, both tumor cell lines were coinjected i.v. with either AdExTek or a control virus. Mice coinjected with control virus developed numerous large, well vascularized lung metastases. In contrast, mice coinjected with AdExTek virus developed few, if any, grossly apparent metastases, and histologic examination revealed only small avascular clusters of tumor cells. Administration of AdExTek also inhibited tumor metastasis when delivered at the time of surgical excision of primary tumors in a clinically relevant model of tumor metastasis. This study demonstrates the potential utility of gene therapy for systemic delivery of an antiangiogenic agent targeting an endothelium-specific receptor, Tie2.
Mineralocorticoid receptor knockout mice: Pathophysiology of Na
+
metabolism
Authors: Berger Stefan , Bleich Markus , Schmid Wolfgang , Cole Timothy J. , Peters Jörg , Watanabe Haruko , Kriz Wilhelm , Warth Richard , Greger Rainer , Schütz Günther
Published: July 2002

Mineralocorticoid receptor (MR)-deficient mice were generated by gene targeting. These animals had a normal prenatal development. During the first week of life, MR-deficient (−/−) mice developed symptoms of pseudohypoaldosteronism. They finally lost weight and eventually died at around day 10 after birth from dehydration by renal sodium and water loss. At day 8, −/− mice showed hyperkalemia, hyponatremia, and a strong increase in renin, angiotensin II, and aldosterone plasma concentrations. Methods were established to measure renal clearance and colonic transepithelial Na
+
reabsorption in 8-day-old mice
in vivo
. The fractional renal Na
+
excretion was elevated >8-fold. The glomerular filtration rate in −/− mice was not different from controls. The effect of amiloride on renal Na
+
excretion and colonic transepithelial voltage reflects the function of amiloide-sensitive epithelial Na
+
channels (ENaC). In −/− mice, it was reduced to 24% in the kidney and to 16% in the colon. There was, however, still significant residual ENaC-mediated Na
+
reabsorption in both epithelia. RNase protection analysis of the subunits of ENaC and (Na
+
+ K
+
)-ATPase did not reveal a decrease in −/− mice. The present data indicate that MR-deficient neonates die because they are not able to compensate renal Na
+
loss. Regulation of Na
+
reabsorption via MR is not achieved by transcriptional control of ENaC and (Na
+
+ K
+
)-ATPase in RNA abundance but by transcriptional control of other as yet unidentified genes. MR knockout mice will be a suitable tool for the search of these genes.
Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells
Authors: O’Rourke Donald M. , Kao Gary D. , Singh Natasha , Park Byeong-Woo , Muschel Ruth J. , Wu Chuan-jin , Greene Mark I.
Published: July 2002

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.
Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes
Authors: Spang Anne , Matsuoka Ken , Hamamoto Susan , Schekman Randy , Orci Lelio
Published: July 2002

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large (≈300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5′-[γ-thio]triphosphate. Coated vesicles are 40–70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5′-[γ-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.
gas7
: A gene expressed preferentially in growth-arrested fibroblasts and terminally differentiated Purkinje neurons affects neurite formation
Authors: Ju Yu-Ten , Chang Annie C. Y. , She Bin-Ru , Tsaur Meei-Ling , Hwang Hwa-Min , Chao Chuck C.-K. , Cohen Stanley N. , Lin-Chao Sue
Published: July 2002

Growth arrest-specific
(gas)
genes are expressed preferentially in cells that enter a quiescent state.
gas7
, which we identified in serum-starved murine fibroblasts, is reported here to be expressed
in vivo
selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum.
gas7
transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using
in situ
hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. Collectively, our results provide evidence for an association between expression of this
gas
gene and neuronal development.
A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase
Authors: Wrobel John A. , Chao Shih-Fong , Conrad Michael J. , Merker Jason D. , Swanstrom Ronald , Pielak Gary J. , Hutchison Clyde A.
Published: July 2002

By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95–203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in
Escherichia coli
, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a “genetic” means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior. In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general. This strategy should be useful for studying proteins for which no crystallographic data are available.
Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein
Authors: Reddy Thipparthi Raghavendar , Li Xinqiang , Jones Ying , Ellisman Mark H. , Ching Gee Y. , Liem Ronald K. H. , Wong-Staal Flossie
Published: July 2002

The human T-cell leukemia virus (HTLV) is associated with adult T cell leukemia and neurological disorders (TSP/HAM). The HTLV transcriptional transactivator, Tax, is known to exert its effect through protein–protein interaction with several transcription factors that activate genes in T cell proliferation. The pathogenic mechanism in the CNS is less defined. Using the yeast two-hybrid system, we have identified a specific Tax-binding protein as the neuronal specific intermediate filament protein, α-internexin. Tax binds to the domain corresponding to the rod region of α-internexin, which is essential for neurofilament assembly. The Tax domains involved in binding are separable from those involved in transactivation. TxBP-1/α-internexin and Tax are expressed in the cytoplasm and nucleus, respectively, when expressed alone, but in coexpressing cells, colocalization of both proteins was observed in a perinuclear, punctate distribution. This
in vivo
interaction also resulted in a dramatic reduction in Tax transactivation and the network formation by α-internexin. The specific interaction of Tax and a neuronal specific intermediate filament protein may provide a clue to the pathogenesis of TSP/HAM.
Distinct steady–state nuclear receptor coregulator complexes exist
in
vivo
Authors: McKenna Neil J. , Nawaz Zafar , Tsai Sophia Y. , Tsai Ming-Jer , O’Malley Bert W.
Published: July 2002

Transcriptional regulation by members of the nuclear hormone receptor superfamily is a modular process requiring the mediation of distinct subclasses of coregulators. These subclasses include members of the steroid receptor coactivator-1 (SRC-1) coactivator family, p300/CBP and their associated proteins, such as p300/CBP-associated factor, human homologs of SWI/SNF proteins such as BRG-1, and the less well-characterized E3 ubiquitin-protein ligases E6 papillomavirus protein-associated protein and receptor-potentiating factor-1. Because functional studies indicate that these coregulators may form higher order complexes, we analyzed steady–state complexes of different coregulator subclasses
in vivo
. T47D and HeLa cell lysates were subjected to biochemical fractionation and screened by immunoblotting using coregulator-specific antibodies. We show that different subclasses of nuclear receptor coregulators exhibit distinct fractionation profiles. Furthermore, evidence is provided that SRC-1 family members may exist
in vivo
in heteromultimeric forms with each other. In addition, we demonstrate that liganded PR is present in stable complexes containing SRC-1 and transcription intermediary factor 2 (TIF2)
in vivo
. Our results suggest that the assembly of large, modular transcriptional complexes by recruitment of distinct subclasses of preformed coregulator subcomplexes may be involved in transcriptional regulation by activated nuclear receptors.
Interpreting functional imaging studies in terms of neurotransmitter cycling
Authors: Shulman Robert G. , Rothman Douglas L.
Published: July 2002

Functional imaging experiments, in particular positron-emission tomography and functional magnetic resonance imaging, can be analyzed either in psychological terms or on the basis of neuroscience. In the usual psychological interpretation, stimulations are designed to activate specific mental processes identified by cognitive psychology, which are then localized by the signals in functional imaging experiments. An alternate approach would be to analyze experiments in terms of the neurobiological processes responsible for the signals. Recent
in vivo
13
C NMR measurements of the glutamate-to-glutamine neurotransmitter cycling in rat and human brains facilitate a neuroscientific interpretation of functional imaging data in terms of neurobiological processes since incremental neurotransmitter flux showed a 1:1 stoichiometry with the incremental rate of glucose oxidation. Because functional imaging signals depend on brain energy consumption, a quantitative relationship can be established between the signal (
S
) and the specific neurochemical cerebral neurotransmitter activity (
N
) of glutamate-to-glutamine neurotransmitter cycling. The quantitation of neuronal activity proposed has implications for the psychological design and interpretation of functional imaging experiments. Measurements of the neurotransmitter cycling flux at rest in functional imaging experiments suggest that performing cognitive tasks and sensory stimulations increases neurotransmitter cycling by only 10–20%. Therefore it cannot be assumed that reference state activities are negligible, nor that they are constant during stimulation.
Human milk lactoferrin inactivates two putative colonization factors expressed by
Haemophilus influenzae
Authors: Qiu Jiazhou , Hendrixson David R. , Baker Edward N. , Murphy Timothy F. , Geme Joseph W. St. , Plaut Andrew G.
Published: July 2002

Haemophilus influenzae
is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against
H. influenzae
colonization and disease. In the present study, we examined the effect of human milk on the
H. influenzae
IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the
H. influenzae
P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of
H. influenzae
by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.
HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter
Authors: Marzio Giuseppe , Tyagi Mudit , Gutierrez Maria Ines , Giacca Mauro
Published: July 2002

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both
in vitro
and
in vivo
. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.
Modeling the impact of global tuberculosis control strategies
Authors: Murray Christopher J. L. , Salomon Joshua A.
Published: July 2002

An epidemiological model of tuberculosis has been developed and applied to five regions of the world. Globally, 6.7 million new cases of tuberculosis and 2.4 million deaths from tuberculosis are estimated for 1998. Based on current trends in uptake of the World Health Organization’s strategy of directly observed treatment, short-course, we expect a total of 225 million new cases and 79 million deaths from tuberculosis between 1998 and 2030. Active case-finding by using mass miniature radiography could save 23 million lives over this period. A single contact treatment for tuberculosis could avert 24 million cases and 11 million deaths; combined with active screening, it could reduce mortality by nearly 40%. A new vaccine with 50% efficacy could lower incidence by 36 million cases and mortality by 9 million deaths. Support for major extensions to global tuberculosis control strategies will occur only if the size of the problem and the potential for action are recognized more widely.
Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: Possible role in antigen-driven reactions in the absence of germinal centers
Authors: Diaz Marilyn , Greenberg Andrew S. , Flajnik Martin F.
Published: July 2002

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark andXenopusIg. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.
Mature monocytic cells enter tissues and engraft
Authors: Kennedy David W. , Abkowitz Janis L.
Published: July 2002

The goal of this study was to identify the circulating cell that is the immediate precursor of tissue macrophages. ROSA 26 marrow mononuclear cells (containing the β-geo transgene that encodes β-galactosidase and neomycin resistance activities) were cultured in the presence of macrophage colony-stimulating factor and flt3 Ligand for 6 days to generate monocytic cells at all stages of maturation. Expanded monocyte cells (EMC), the immature (ER-MP12
+
) and more mature (ER-MP20
+
) subpopulations, were transplanted into irradiated B6/129 F2 mice. β-gal staining of tissue sections from animals 15 min after transplantation demonstrated that the donor cells landed randomly. By 3 h, donor cells in lung and liver were more frequent in animals transplanted with ER-MP20
+
(more mature) EMC than in animals transplanted with unseparated EMC or fresh marrow mononuclear cells, a pattern that persisted at 3 and 7 days. At 3 days, donor cells were found in spleen, liver, lung, and brain (rarely) as clusters as well as individual cells. By 7 and 14 days, the clusters had increased in size, and the cells expressed the macrophage antigen F4/80, suggesting that further replication and differentiation had occurred. PCR for the neogene was used to quantitate the amount of donor DNA in tissues from transplanted animals and confirmed that ER-MP20
+
EMC preferentially engrafted. These data demonstrate that a mature monocytic cell gives rise to tissue macrophages. Because these cells can be expanded and manipulated
in vitro
, they may be a suitable target population for gene therapy of lysosomal storage diseases.
Production of β-defensins by human airway epithelia
Authors: Singh Pradeep K. , Jia Hong Peng , Wiles Kerry , Hesselberth Jay , Liu Lide , Conway Barbara-Ann D. , Greenberg Everett P. , Valore Erika V. , Welsh Michael J. , Ganz Tomas , Tack Brian F. , McCray Paul B.
Published: July 2002

Human β-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1β stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.
Functional neuroimaging studies of encoding, priming, and explicit memory retrieval
Authors: Buckner Randy L. , Koutstaal Wilma
Published: July 2002

Human functional neuroimaging techniques provide a powerful means
of linking neural level descriptions of brain function and cognition.
The exploration of the functional anatomy underlying human memory
comprises a prime example. Three highly reliable findings linking
memory-related cognitive processes to brain activity are discussed.
First, priming is accompanied by reductions in the amount of neural
activation relative to naive or unprimed task performance. These
reductions can be shown to be both anatomically and functionally
specific and are found for both perceptual and conceptual task
components. Second, verbal encoding, allowing subsequent conscious
retrieval, is associated with activation of higher order brain regions
including areas within the left inferior and dorsal prefrontal cortex.
These areas also are activated by working memory and effortful word
generation tasks, suggesting that these tasks, often discussed as
separable, might rely on interdependent processes. Finally, explicit
(intentional) retrieval shares much of the same functional anatomy as
the encoding and word generation tasks but is associated with the
recruitment of additional brain areas, including the anterior
prefrontal cortex (right > left). These findings illustrate how
neuroimaging techniques can be used to study memory processes and can
both complement and extend data derived through other means. More
recently developed methods, such as event-related functional MRI, will
continue this progress and may provide additional new directions for
research.
Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase
Authors: Van Hellemond Jaap J. , Opperdoes Fred R. , Tielens Aloysius G. M.
Published: July 2002

Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same α-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes ofLeishmania mexicana mexicana,L. infantumandPhytomonassp., and procyclics ofTrypanosoma bruceiwere analyzed as well as the parasitic helminthFasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT ofL. mexicana mexicanapromastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km0.26 mM) and a low affinity for succinate (Km6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles.
Functions of the two glutamate transporters GLAST and GLT-1 in the retina
Authors: Harada Takayuki , Harada Chikako , Watanabe Masahiko , Inoue Yoshiro , Sakagawa Takashi , Nakayama Naoki , Sasaki Shoji , Okuyama Shigeru , Watase Kei , Wada Keiji , Tanaka Kohichi
Published: July 2002

In the retina, the glutamate transporter GLAST is expressed in
Müller cells, whereas the glutamate transporter GLT-1 is found
only in cones and various types of bipolar cells. To investigate the
functional role of this differential distribution of glutamate
transporters, we have analyzed GLAST and GLT-1 mutant mice. In
GLAST-deficient mice, the electroretinogram b-wave and oscillatory
potentials are reduced and retinal damage after ischemia is
exacerbated, whereas GLT-1-deficient mice show almost normal
electroretinograms and mild increased retinal damage after ischemia.
These results demonstrate that GLAST is required for normal signal
transmission between photoreceptors and bipolar cells and that both
GLAST and GLT-1 play a neuroprotective role during ischemia in the
retina.
Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats
Authors: Suzuki Hidekazu , DeLano Frank A. , Parks Dale A. , Jamshidi Neema , Granger D. Neil , Ishii Hiromasa , Suematsu Makoto , Zweifach Benjamin W. , Schmid-Schönbein Geert W.
Published: July 2002

Recent evidencein vivoindicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (−)BOF4272 [(−)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-α)-1,3,5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was ≈3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (−)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.
Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development
Authors: Arman Esther , Haffner-Krausz Rebecca , Chen Yali , Heath John K. , Lonai Peter
Published: July 2002

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a
neo
cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells
in vitro
, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.
Genes controlling fertilization-independent seed development in
Arabidopsis thaliana
Authors: Luo Ming , Bilodeau Pierre , Koltunow Anna , Dennis Elizabeth S. , Peacock W. James , Chaudhury Abdul M.
Published: July 2002

We have cloned two genes,
FIS1
and
FIS2
, that control both fertilization independent seed
development and postpollination embryo development in
Arabidopsis
. These genes confer female gametophytic
phenotypes.
FIS2
encodes a protein with a
C
2
H
2
zinc-finger motif and three putative
nuclear localization signals, indicating that it is likely to be a
transcription factor.
FIS1
encodes a protein with
homology to the
Drosophila Polycomb
group gene
Enhancer-of-zeste
and is identical to the recently
described
Arabidopsis
gene
MEDEA
. FIS1 is
a protein with a number of putative functional domains, including the
SET domain present in Enhancer-of-zeste-related proteins. Comparison of
the position of the lesions in the
fis1
and
medea
mutant alleles indicates that
fis1
is a null allele producing a truncated polypeptide lacking all the
protein domains whereas the deduced protein from
medea
lacks only the SET domain. We present a model of the role of
FIS1
and
FIS2
gene products in seed
development.
A high-density genome scan detects evidence for a bipolar-disorder susceptibility locus on 13q32 and other potential loci on 1q32 and 18p11.2
Authors: Detera-Wadleigh Sevilla D. , Badner Judith A. , Berrettini Wade H. , Yoshikawa Takeo , Goldin Lynn R. , Turner Gordon , Rollins Denise Y. , Moses Tracy , Sanders Alan R. , Karkera Jayaprakash D. , Esterling Lisa E. , Zeng Jin , Ferraro Thomas N. , Guroff Juliet J. , Kazuba Diane , Maxwell Mary E. , Nurnberger John I. , Gershon Elliot S.
Published: July 2002

Bipolar disorder is a severe mental illness characterized by mood swings of elation and depression. Family, twin, and adoption studies suggest a complex genetic etiology that may involve multiple susceptibility genes and an environmental component. To identify chromosomal loci contributing to vulnerability, we have conducted a genome-wide scan on ≈396 individuals from 22 multiplex pedigrees by using 607 microsatellite markers. Multipoint nonparametric analysis detected the strongest evidence for linkage at 13q32 with a maximal logarithm of odds (lod) score of 3.5 (
P
= 0.000028) under a phenotype model that included bipolar I, bipolar II with major depression, schizoaffective disorder, and recurrent unipolar disorder. Suggestive linkage was found on 1q31-q32 (lod = 2.67;
P
= 0.00022) and 18p11.2 (lod = 2.32;
P
= 0.00054). Recent reports have linked schizophrenia to 13q32 and 18p11.2. Our genome scan identified other interesting regions, 7q31 (lod = 2.08;
P
= 0.00099) and 22q11-q13 (lod = 2.1;
P
= 0.00094), and also confirmed reported linkages on 4p16, 12q23-q24, and 21q22. By comprehensive screening of the entire genome, we detected unreported loci for bipolar disorder, found support for proposed linkages, and gained evidence for the overlap of susceptibility regions for bipolar disorder and schizophrenia.
Mutations in
HYAL1
, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX
Authors: Triggs-Raine Barbara , Salo Timothy J. , Zhang Hong , Wicklow Brandy A. , Natowicz Marvin R.
Published: July 2002

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes,
HYAL1
and
HYAL2
, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the
HYAL1
alleles of the patient, a 1412G → A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene,
HYAL3
, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.
Ion channels gated by heat
Authors: Cesare P. , Moriondo A. , Vellani V. , McNaughton P. A.
Published: July 2002

All animals need to sense temperature to avoid hostile environments
and to regulate their internal homeostasis. A particularly obvious
example is that animals need to avoid damagingly hot stimuli. The
mechanisms by which temperature is sensed have until recently been
mysterious, but in the last couple of years, we have begun to
understand how noxious thermal stimuli are detected by sensory neurons.
Heat has been found to open a nonselective cation channel in primary
sensory neurons, probably by a direct action. In a separate study, an
ion channel gated by capsaicin, the active ingredient of chili peppers,
was cloned from sensory neurons. This channel (vanilloid receptor
subtype 1, VR1) is gated by heat in a manner similar to the native
heat-activated channel, and our current best guess is that this channel
is the molecular substrate for the detection of painful heat. Both the
heat channel and VR1 are modulated in interesting ways. The response of
the heat channel is potentiated by phosphorylation by protein kinase C,
whereas VR1 is potentiated by externally applied protons. Protein
kinase C is known to be activated by a variety of inflammatory
mediators, including bradykinin, whereas extracellular acidification is
characteristically produced by anoxia and inflammation. Both modulatory
pathways are likely, therefore, to have important physiological
correlates in terms of the enhanced pain (hyperalgesia) produced by
tissue damage and inflammation. Future work should focus on
establishing, in molecular terms, how a single ion channel can detect
heat and how the detection threshold can be modulated by hyperalgesic
stimuli.
Right hemispheric dominance of inhibitory control: An event-related functional MRI study
Authors: Garavan H. , Ross T. J. , Stein E. A.
Published: July 2002

Normal human behavior and cognition are reliant on a person’s ability to inhibit inappropriate thoughts, impulses, and actions. The temporal and spatial advantages of event-related functional MRI (fMRI) were exploited to identify cortical regions that showed a transient change in fMRI signal after the withholding of a prepotent motor response. The temporal specificity of the event-related fMRI design also minimized possible contamination from response inhibition errors (i. e., commission errors) and other extraneous processes. Regions identified were strongly lateralized to the right hemisphere and included the middle and inferior frontal gyri, frontal limbic area, anterior insula, and inferior parietal lobe. Contrary to the prominence traditionally given to ventral frontal regions for response inhibition, the results suggest that response inhibition is accomplished by a distributed cortical network.
Radiation hybrid mapping of the zebrafish genome
Authors: Hukriede Neil A. , Joly Lucille , Tsang Michael , Miles Jennifer , Tellis Patricia , Epstein Jonathan A. , Barbazuk William B. , Li Frank N. , Paw Barry , Postlethwait John H. , Hudson Thomas J. , Zon Leonard I. , McPherson John D. , Chevrette Mario , Dawid Igor B. , Johnson Stephen L. , Ekker Marc
Published: July 2002

The zebrafish is an excellent genetic system for the study of vertebrate development and disease. In an effort to provide a rapid and robust tool for zebrafish gene mapping, a panel of radiation hybrids (RH) was produced by fusion of irradiated zebrafish AB9 cells with mouse B78 cells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the LN54 panel is 22%. Characterization of the LN54 panel with 849 simple sequence length polymorphism markers, 84 cloned genes and 122 expressed sequence tags allowed the production of an RH map whose total size was 11,501 centiRays. From this value, we estimated the average breakpoint frequency of the LN54 RH panel to correspond to 1 centiRay = 148 kilobase. Placement of a group of 235 unbiased markers on the RH map suggests that the map generated for the LN54 panel, at present, covers 88% of the zebrafish genome. Comparison of marker positions in RH and meiotic maps indicated a 96% concordance. Mapping expressed sequence tags and cloned genes by using the LN54 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish.
The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin
Authors: Weinreich Michael , Liang Chun , Stillman Bruce
Published: July 2002

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6
K114E
protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.
Calibrating bacterial evolution
Authors: Ochman Howard , Elwyn Susannah , Moran Nancy A.
Published: July 2002

Attempts to calibrate bacterial evolution have relied on the assumption that rates of molecular sequence divergence in bacteria are similar to those of higher eukaryotes, or to those of the few bacterial taxa for which ancestors can be reliably dated from ecological or geological evidence. Despite similarities in the substitution rates estimated for some lineages, comparisons of the relative rates of evolution at different classes of nucleotide sites indicate no basis for their universal application to all bacteria. However, there is evidence that bacteria have a constant genome-wide mutation rate on an evolutionary time scale but that this rate differs dramatically from the rate estimated by experimental methods.
Phase variation of the
lpf
operon is a mechanism to evade cross-immunity between
Salmonella
serotypes
Authors: Norris Tracy L. , Bäumler Andreas J.
Published: July 2002

Conventional wisdom holds that phase variation is a mechanism for immune evasion. However, despite fimbrial phase variation, mice previously exposed to
Salmonella typhimurium
are protected against a subsequent challenge. We evaluated whether
lpf
phase variation instead may be a mechanism to evade cross-immunity between
Salmonella
serotypes. Mice were immunized orally with
S. typhimurium aroA
mutants either that expressed the
lpf
operon (phase-on variant) or in which the entire
lpf
operon had been removed by deletion. During a subsequent challenge with virulent
Salmonella enteritidis
a selection against
lpf
phase-on variants was observed in mice previously exposed to
S. typhimurium
long polar fimbriae. Vaccination with
S. typhimurium
did not confer protection against challenge with
S. enteritidis
, presumably because
lpf
phase-off variants were able to evade cross-immunity. We propose that
lpf
phase variation is a mechanism to evade cross-immunity between
Salmonella
serotypes, thereby allowing their coexistence in a host population.
Arabidopsis thaliana PAD4
encodes a lipase-like gene that is important for salicylic acid signaling
Authors: Jirage Dayadevi , Tootle Tina L. , Reuber T. Lynne , Frost Louise N. , Feys Bart J. , Parker Jane E. , Ausubel Frederick M. , Glazebrook Jane
Published: July 2002

The Arabidopsis
PAD4
gene previously was found to be required for expression of multiple defense responses including camalexin synthesis and
PR-1
gene expression in response to infection by the bacterial pathogen
Pseudomonas syringae
pv.
maculicola.
This report describes the isolation of
PAD4
. The predicted PAD4 protein sequence displays similarity to triacyl glycerol lipases and other esterases. The
PAD4
transcript was found to accumulate after
P. syringae
infection or treatment with salicylic acid (SA).
PAD4
transcript levels were very low in infected
pad4
mutants. Treatment with SA induced expression of
PAD4
mRNA in
pad4–1
,
pad4–3
, and
pad4–4
plants but not in
pad4–2
plants. Induction of
PAD4
expression by
P. syringae
was independent of the regulatory factor NPR1 but induction by SA was NPR1-dependent. Taken together with the previous observation that
pad4
mutants have a defect in accumulation of SA upon pathogen infection, these results suggest that PAD4 participates in a positive regulatory loop that increases SA levels, thereby activating SA-dependent defense responses.
Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages
Authors: Underhill David M. , Ozinsky Adrian , Smith Kelly D. , Aderem Alan
Published: July 2002

The recognition of mycobacterial cell wall components causes macrophages to secrete tumor necrosis factor α (TNF-α) and other cytokines that are essential for the development of a protective inflammatory response. We show that toll-like receptors are required for the induction of TNF-α in macrophages byMycobacterium tuberculosis. Expression of a dominant negative form of MyD88 (a signaling component required for toll-like receptor signaling) in a mouse macrophage cell line blocks TNF-α production induced byM. tuberculosis. We identify toll-like receptor-2 (TLR2) as the specific toll-like receptor required for this induction by showing that expression of an inhibitory TLR2 (TLR2-P681H) blocks TNF-α production induced by wholeM. tuberculosis. Further, we show that TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarrabinomannan, mycolylarabinogalactan–peptidoglycan complex, orM. tuberculosistotal lipids. Thus, although many mycobacterial cell wall fractions are identified to be inflammatory, all require TLR2 for induction of TNF-α in macrophages. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria.