bluuebunny/crossref_metadata_2025 huggingface dataset:
Precise gene localization by phenotypic assay of radiation hybrid cells
Authors: Rasko John E. J. , Battini Jean-Luc , Kruglyak Leonid , Cox David R. , Miller A. Dusty
Published: July 2002

A high resolution map of the human genome previously has been constructed by using the G3 panel of human/hamster radiation hybrid cell lines and >15,000 unique human genetic markers. By determining whether human DNA sequences are present or absent in each of the hybrids, localization of single genes may routinely be achieved at ≈250-kb resolution. In this paper we have tested whether similarly precise localization might be achieved by phenotypic screening of the hybrids to facilitate positional cloning of unknown genes. We assayed the susceptibility of each of the hybrid cell lines to transduction by retroviral vectors bearing different retroviral envelope proteins that recognize receptors present on human but not on hamster cells. The results for each of the retroviral vectors were informative and allowed precise localization of the receptor genes for the RD114 cat endogenous retrovirus, xenotropic murine leukemia virus, and type C feline leukemia virus. After cloning of the receptors for these retroviruses, we found that standard genotypic mapping by PCR gave results that were nearly identical to those from phenotypic mapping. These experiments show that precise gene localization by phenotypic assay of radiation hybrids is practical and was not appreciably impacted by the known instability of such hybrid cells. This technique should be applicable to many other human genes having discernible phenotypes in hamster cells and, with completion of the human genome project, will allow rapid identification of unknown genes on the basis of phenotype.
Multiple transcription-factor genes are early targets of phytochrome A signaling
Authors: Tepperman James M. , Zhu Tong , Chang Hur-Song , Wang Xun , Quail Peter H.
Published: July 2002

The phytochrome family of sensory photoreceptors directs adaptational changes in gene expression in response to environmental light signals. Using oligonucleotide microarrays to measure expression profiles in wild-type and phytochrome A (phyA) null-mutant
Arabidopsis
seedlings, we have shown that 10% of the genes represented on the array are regulated by phyA in response to a continuous far-red light signal. Strikingly, 44% of the genes responding to the signal within 1 h are predicted to encode multiple classes of transcriptional regulators. Together with previous data, this observation suggests that phyA may regulate seedling photomorphogenesis by direct targeting of light signals to the promoters of genes encoding a master set of diverse transcriptional regulators, responsible in turn for orchestrating the expression of multiple downstream target genes in various branches of a phyA-regulated transcriptional network.
Three-dimensional structure of a mammalian thioredoxin reductase: Implications for mechanism and evolution of a selenocysteine-dependent enzyme
Authors: Sandalova Tatyana , Zhong Liangwei , Lindqvist Ylva , Holmgren Arne , Schneider Gunter
Published: July 2002

Thioredoxin reductases (TrxRs) from mammalian cells contain an
essential selenocysteine residue in the conserved C-terminal sequence
Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme.
Reduction by NADPH generates a selenolthiol, which is the active site
in reduction of Trx. The three-dimensional structure of the SeCys498Cys
mutant of rat TrxR in complex with NADP
+
has been
determined to 3.0-Å resolution by x-ray crystallography. The overall
structure is similar to that of glutathione reductase (GR), including
conserved amino acid residues binding the cofactors FAD and NADPH.
Surprisingly, all residues directly interacting with the substrate
glutathione disulfide in GR are conserved despite the failure of
glutathione disulfide to act as a substrate for TrxR. The 16-residue
C-terminal tail, which is unique to mammalian TrxR, folds in such a way
that it can approach the active site disulfide of the other subunit in
the dimer. A model of the complex of TrxR with Trx suggests that
electron transfer from NADPH to the disulfide of the substrate is
possible without large conformational changes. The C-terminal extension
typical of mammalian TrxRs has two functions: (
i
) it
extends the electron transport chain from the catalytic disulfide to
the enzyme surface, where it can react with Trx, and
(
ii
) it prevents the enzyme from acting as a GR by
blocking the redox-active disulfide. Our results suggest that mammalian
TrxR evolved from the GR scaffold rather than from its prokaryotic
counterpart. This evolutionary switch renders cell growth dependent on
selenium.
A common polymorphism associated with antibiotic-induced cardiac arrhythmia
Authors: Sesti Federico , Abbott Geoffrey W. , Wei Jian , Murray Katherine T. , Saksena Sanjeev , Schwartz Peter J. , Priori Silvia G. , Roden Dan M. , George Alfred L. , Goldstein Steve A. N.
Published: July 2002

Drug-induced long QT syndrome (LQTS) is a prevalent disorder of uncertain etiology that predisposes to sudden death.
KCNE2
encodes MinK-related peptide 1 (MiRP1), a subunit of the cardiac potassium channel I
Kr
that has been associated previously with inherited LQTS. Here, we examine
KCNE2
in 98 patients with drug-induced LQTS, identifying three individuals with sporadic mutations and a patient with sulfamethoxazole-associated LQTS who carried a single-nucleotide polymorphism (SNP) found in ≈1.6% of the general population. While mutant channels showed diminished potassium flux at baseline and wild-type drug sensitivity, channels with the SNP were normal at baseline but inhibited by sulfamethoxazole at therapeutic levels that did not affect wild-type channels. We conclude that allelic variants of MiRP1 contribute to a significant fraction of cases of drug-induced LQTS through multiple mechanisms and that common sequence variations that increase the risk of life-threatening drug reactions can be clinically silent before drug exposure.
A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus
Authors: Mayo Lindsey D. , Donner David B.
Published: July 2002

The Mdm2 oncoprotein promotes cell survival and cell cycle
progression by inhibiting the p53 tumor suppressor protein. To regulate
p53, Mdm2 must gain nuclear entry, and the mechanism that induces this
is now identified. Mitogen-induced activation of phosphatidylinositol
3-kinase (PI3-kinase) and its downstream target, the Akt/PKB
serine-threonine kinase, results in phosphorylation of Mdm2 on serine
166 and serine 186. Phosphorylation on these sites is necessary for
translocation of Mdm2 from the cytoplasm into the nucleus.
Pharmacological blockade of PI3-kinase/Akt signaling or expression of
dominant-negative PI3-kinase or Akt inhibits nuclear entry of Mdm2,
increases cellular levels of p53, and augments p53 transcriptional
activity. Expression of constitutively active Akt promotes nuclear
entry of Mdm2, diminishes cellular levels of p53, and decreases p53
transcriptional activity. Mutation of the Akt phosphorylation sites in
Mdm2 produces a mutant protein that is unable to enter the nucleus and
increases p53 activity. The demonstration that PI3-kinase/Akt
signaling affects Mdm2 localization provides insight into how this
pathway, which is inappropriately activated in many malignancies,
affects the function of p53.
Focal adhesion kinase is involved in mechanosensing during fibroblast migration
Authors: Wang Hong-Bei , Dembo Micah , Hanks Steven K. , Wang Yu-li
Published: July 2002

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine
kinase localized at focal adhesions and is believed to mediate
adhesion-stimulated effects. Although ablation of FAK impairs cell
movement, it is not clear whether FAK might be involved in the guidance
of cell migration, a role consistent with its putative regulatory
function. We have transfected FAK-null fibroblasts with FAK gene under
the control of the tetracycline repression system. Cells were cultured
on flexible polyacrylamide substrates for the detection of traction
forces and the application of mechanical stimulation. Compared with
control cells expressing wild-type FAK, FAK-null cells showed a
decrease in migration speed and directional persistence. In addition,
whereas FAK-expressing cells responded to exerted forces by reorienting
their movements and forming prominent focal adhesions, FAK-null cells
failed to show such responses. Furthermore, FAK-null cells showed
impaired responses to decreases in substrate flexibility, which causes
control cells to generate weaker traction forces and migrate away from
soft substrates. Cells expressing Y397F FAK, which cannot be
phosphorylated at a key tyrosine site, showed similar defects in
migration pattern and force-induced reorientation as did FAK-null
cells. However, other aspects of F397-FAK cells, including the
responses to substrate flexibility and the amplification of focal
adhesions upon mechanical stimulation, were similar to that of control
cells. Our results suggest that FAK plays an important role in the
response of migrating cells to mechanical input. In addition,
phosphorylation at Tyr-397 is required for some, but not all, of the
functions of FAK in cell migration.
The visual paired-comparison task as a measure of declarative memory
Authors: Manns Joseph R. , Stark Craig E. L. , Squire Larry R.
Published: July 2002

Performance on the visual paired-comparison task depends on the
integrity of the hippocampal formation in humans, monkeys, and, for an
analogous task, in rats. The present study sought additional evidence
in healthy volunteers concerning the nature of this task. We found that
performance on the visual paired-comparison task was predictive of
subsequent recognition memory performance whereas perceptual priming
was unrelated to subsequent recognition memory performance. The results
are consistent with the data from lesions and suggest that performance
on the visual paired-comparison task measures a form of declarative
memory.
Structure of trichodiene synthase from
Fusarium sporotrichioides
provides mechanistic inferences on the terpene cyclization cascade
Authors: Rynkiewicz Michael J. , Cane David E. , Christianson David W.
Published: July 2002

The x-ray crystal structure of recombinant trichodiene synthase from
Fusarium sporotrichioides
has been determined to 2.5-Å resolution, both unliganded and complexed with inorganic pyrophosphate. This reaction product coordinates to three Mg
2+
ions near the mouth of the active site cleft. A comparison of the liganded and unliganded structures reveals a ligand-induced conformational change that closes the mouth of the active site cleft. Binding of the substrate farnesyl diphosphate similarly may trigger this conformational change, which would facilitate catalysis by protecting reactive carbocationic intermediates in the cyclization cascade. Trichodiene synthase also shares significant structural similarity with other sesquiterpene synthases despite a lack of significant sequence identity. This similarity indicates divergence from a common ancestor early in the evolution of terpene biosynthesis.
Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the
Drosophila
genome
Authors: Berman Benjamin P. , Nibu Yutaka , Pfeiffer Barret D. , Tomancak Pavel , Celniker Susan E. , Levine Michael , Rubin Gerald M. , Eisen Michael B.
Published: July 2002

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early
Drosophila
embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene
giant (gt)
, and show that it acts as an enhancer that recapitulates the posterior expression pattern of
gt.
The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics
Authors: Long Stephen B. , Hancock Paula J. , Kral Astrid M. , Hellinga Homme W. , Beese Lorena S.
Published: July 2002

Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ras superfamily. Farnesylation is essential both for normal functioning of these proteins, and for the transforming activity of oncogenic mutants. Consequently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatment. Here, we present the crystal structure of human FTase, as well as ternary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tetrapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (FPP), or a nonreactive analogue. These structures reveal the structural mechanism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnesylated, and are more effective inhibitors than farnesylated CaaX tetrapeptides. CVFM and L-739,750 are not farnesylated, because these inhibitors bind in a conformation that is distinct from the TKCVFM hexapeptide substrate. This non-substrate binding mode is stabilized by an ion pair between the peptide N terminus and the α-phosphate of the FPP substrate. Conformational mapping calculations reveal the basis for the sequence specificity in the third position of the CaaX motif that determines whether a tetrapeptide is a substrate or non-substrate. The presence of β-branched amino acids in this position prevents formation of the non-substrate conformation; all other aliphatic amino acids in this position are predicted to form the non-substrate conformation, provided their N terminus is available to bind to the FPP α-phosphate. These results may facilitate further development of FTase inhibitors.
A genome-wide analysis of immune responses in
Drosophila
Authors: Irving Phil , Troxler Laurent , Heuer Timothy S. , Belvin Marcia , Kopczynski Casey , Reichhart Jean-Marc , Hoffmann Jules A. , Hetru Charles
Published: July 2002

Oligonucleotide DNA microarrays were used for a genome-wide
analysis of immune-challenged
Drosophila
infected with
Gram-positive or Gram-negative bacteria, or with fungi. Aside from the
expression of an established set of immune defense genes, a significant
number of previously unseen immune-induced genes were found.
Genes of particular interest include
corin
- and
Stubble
-like genes, both of which have a type II
transmembrane domain;
easter
- and
snake
-like genes, which may fulfil the roles of
easter
and
snake
in the Toll pathway; and
a
masquerade
-like gene, potentially involved in enzyme
regulation. The microarray data has also helped to greatly reduce the
number of target genes in large gene groups, such as the proteases,
helping to direct the choices for future mutant studies. Many of the
up-regulated genes fit into the current conceptual framework of host
defense, whereas others, including the substantial number of genes with
unknown functions, offer new avenues for research.
Cloning of a novel receptor expressed in rat prostate and ovary.
Authors: Kuiper G G , Enmark E , Pelto-Huikko M , Nilsson S , Gustafsson J A
Published: July 2002

We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.
HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.
Authors: Feng Y X , Copeland T D , Henderson L E , Gorelick R J , Bosche W J , Levin J G , Rein A
Published: July 2002

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.
Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.
Authors: Khan B V , Harrison D G , Olbrych M T , Alexander R W , Medford R M
Published: July 2002

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.
Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.
Authors: Daro E , van der Sluijs P , Galli T , Mellman I
Published: July 2002

During receptor mediated endocytosis, at least a fraction of recycling cargo typically accumulates in a pericentriolar cluster of tubules and vesicles. However, it is not clear if these endosomal structures are biochemically distinct from the early endosomes from which they are derived. To better characterize this pericentriolar endosome population, we determined the distribution of two endogenous proteins known to be functionally involved in receptor recycling [Rab4, cellubrevin (Cbvn)] relative to the distribution of a recycling ligand [transferrin (Tfn)] as it traversed the endocytic pathway. Shortly after internalization, Tfn entered a population of early endosomes that contained both Rab4 and Cbvn, demonstrated by triple label immunofluorescence confocal microscopy. Tfn then accumulated in the pericentriolar cluster of recycling vesicles (RVs). However, although these pericentriolar endosomes contained Cbvn, they were strikingly depleted of Rab4. The ability of internalized Tfn to reach the Rab4-negative population was not blocked by nocodazole, although the characteristic pericentriolar location of the population was not maintained in the absence of microtubules. Similarly, Rab4-positive and -negative populations remained distinct in cells treated with brefeldin A, with only Rab4-positive elements exhibiting the extended tubular morphology induced by the drug. Thus, at least with respect to Rab4 distribution, the pathway of Tfn receptor recycling consists of at least two biochemically and functionally distinct populations of endosomes, a Rab4-positive population of early endosomes to which incoming Tfn is initially delivered and a Rab4-negative population of recycling vesicles that transiently accumulates Tfn on its route back to the plasma membrane.
Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.
Authors: Brill S R , Ross K E , Davidow C J , Ye M , Grantham J J , Caplan M J
Published: July 2002

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.
Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.
Authors: Caruso M , Pham-Nguyen K , Kwong Y L , Xu B , Kosai K I , Finegold M , Woo S L , Chen S H
Published: July 2002

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.
Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.
Authors: Adelroth P , Sigurdson H , Hallén S , Brzezinski P
Published: July 2002

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.
Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.
Authors: Akaike T , Noguchi Y , Ijiri S , Setoguchi K , Suga M , Zheng Y M , Dietzschold B , Maeda H
Published: July 2002

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.
Activity of primate inferotemporal neurons related to a sought target in pair-association task.
Authors: Naya Y , Sakai K , Miyashita Y
Published: July 2002

Visual long-term memory in primates has been assessed by using the pair-association (PA) task, in which a subject retrieves and chooses the paired associate of a cue picture. Our previous studies on single neurons in the anterior inferotemporal (AIT) cortex suggested their roles in representing paired associates in the mind. To test the possibility that the delay activity of AIT neurons is related to a particular picture as a sought target, we devised the PA with color switch (PACS) task. In the PACS task, the necessity for memory retrieval and its initiation time were controlled by a color switch in the middle of the delay period. A control task, in which there is no color switch, corresponds to the conventional delayed matching-to-sample (DMS) task where the monkey chooses the same picture as a cue. We found that AIT neurons started to respond just after the color switch in the PACS task, when the cue-optimal picture's associate was presented as a cue. In contrast, they showed no response change in the DMS task. We confirmed that this effect is not due to the visual response to colors. Furthermore, when the cue-optimal picture was presented as a cue, these neurons showed suppression after the color switch in the PACS task. These results suggest that the activity of AIT neurons mediates gating mechanisms that preferentially pass information about a sought target, even when the sought target is retrieved from long-term memory.
Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation.
Authors: Jurutka P W , Hsieh J C , Nakajima S , Haussler C A , Whitfield G K , Haussler M R
Published: July 2002

The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.
Jasmonate is essential for insect defense in
Arabidopsis
Authors: McConn Michele , Creelman Robert A. , Bell Erin , Mullet John E. , Browse John
Published: July 2002

The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in
Arabidopsis
and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (
fad3–2 fad7–2 fad8
) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality (≈80%) from attack by larvae of a common saprophagous fungal gnat,
Bradysia impatiens
(Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to ≈12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant–insect interaction. The transcripts of three wound-responsive genes were shown not to be induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione
S
-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.
Characterization of the thyroid Na
+
/I
−
symporter with an anti-COOH terminus antibody
Authors: Levy Orlie , Dai Ge , Riedel Claudia , Ginter Christopher S. , Paul Elliot M. , Lebowitz Adam N. , Carrasco Nancy
Published: July 2002

The Na
+
/I
−
symporter (NIS) is the plasma membrane protein that catalyzes active I
−
transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; ≈65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a ≈87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells,
Xenopus laevis
oocytes, and COS cells expressing NIS with peptidyl
N
-glycanase F converted the ≈87 kDa-polypeptide into a ≈50 kDa-species, the same relative molecular weight exhibited by NIS expressed in
E. coli
. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (≈56 kDa) and the mature ≈87 kDa form. Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression
in vivo
was demonstrated.
Impaired fertility in mice deficient for the testicular germ-cell protease PC4
Authors: Mbikay Majambu , Tadros Haidy , Ishida Norito , Lerner Charlie P. , De Lamirande Eve , Chen Andrew , El-Alfy Mohamed , Clermont Yves , Seidah Nabil G. , Chrétien Michel , Gagnon Claude , Simpson Elizabeth M.
Published: July 2002

PC4 is a member of the proprotein convertase family of serine proteases implicated in the processing of a variety of polypeptides including prohormones, proneuropeptides, and cell surface proteins. In rodents, PC4 transcripts have been detected in spermatocytes and round spermatids exclusively, suggesting a reproductive function for this enzyme. In an effort to elucidate this function, we have disrupted its locus (Pcsk4) by homologous recombination in embryonic stem cells and have produced mice carrying the mutation. In intercrosses of heterozygous mutant mice, there was low transmission of the mutantPcsk4allele to the progeny, resulting in lower than expected incidence of heterozygosity and null homozygosity. Thein vivofertility of homozygous mutant males was severely impaired in the absence of any evident spermatogenic abnormality.In vitro, the fertilizing ability ofPcsk4null spermatozoa was also found to be significantly reduced. Moreover, eggs fertilized by these spermatozoa failed to grow to the blastocyst stage. These results suggest that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.
The herpes simplex virus 1 protein kinase U
S
3 is required for protection from apoptosis induced by the virus
Authors: Leopardi Rosario , Van Sant Charles , Roizman Bernard
Published: July 2002

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (α4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled U
S
3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type α4 gene but from which the U
S
3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the U
S
3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the U
S
3 gene as the principal viral product required to block apoptosis.
Abrogation of the alternative complement pathway by targeted deletion of murine factor B
Authors: Matsumoto Mitsuru , Fukuda Wataru , Circolo Antonella , Goellner Joseph , Strauss-Schoenberger Jena , Wang Xuefeng , Fujita Shigeru , Hidvegi Tunde , Chaplin David D. , Colten Harvey R.
Published: July 2002

To investigate the role of complement protein factor B (Bf) and alternative pathway activityin vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with theneorgene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1generation at a frequency suggesting that Bf deficiencyalonehas no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P= 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functionsin vivo.
Excitatory–inhibitory network in the visual cortex: Psychophysical evidence
Authors: Adini Yael , Sagi Dov , Tsodyks Misha
Published: July 2002

At early stages in visual processing cells respond to local stimuli with specific features such as orientation and spatial frequency. Although the receptive fields of these cells have been thought to be local and independent, recent physiological and psychophysical evidence has accumulated, indicating that the cells participate in a rich network of local connections. Thus, these local processing units can integrate information over much larger parts of the visual field; the pattern of their response to a stimulus apparently depends on the context presented. To explore the pattern of lateral interactions in human visual cortex under different context conditions we used a novel chain lateral masking detection paradigm, in which human observers performed a detection task in the presence of different length chains of high-contrast-flanked Gabor signals. The results indicated a nonmonotonic relation of the detection threshold with the number of flankers. Remote flankers had a stronger effect on target detection when the space between them was filled with other flankers, indicating that the detection threshold is caused by dynamics of large neuronal populations in the neocortex, with a major interplay between excitation and inhibition. We considered a model of the primary visual cortex as a network consisting of excitatory and inhibitory cell populations, with both short- and long-range interactions. The model exhibited a behavior similar to the experimental results throughout a range of parameters. Experimental and modeling results indicated that long-range connections play an important role in visual perception, possibly mediating the effects of context.
A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism
Authors: Rigotti Attilio , Trigatti Bernardo L. , Penman Marsha , Rayburn Helen , Herz Joachim , Krieger Monty
Published: July 2002

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (
i
) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (
ii
) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (
iii
) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.
The roles of specific xanthophylls in photoprotection
Authors: Niyogi Krishna K. , Björkman Olle , Grossman Arthur R.
Published: July 2002

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga
Chlamydomonas reinhardtii
revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.
The anaphase inhibitor of
Saccharomyces cerevisiae
Pds1p is a target of the DNA damage checkpoint pathway
Authors: Cohen-Fix Orna , Koshland Doug
Published: July 2002

Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In
Saccharomyces cerevisiae
, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct. We have previously shown that the
S. cerevisiae
protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis.