bluuebunny/crossref_metadata_2025 huggingface dataset:
Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.
Authors: Turki J , Lorenz J N , Green S A , Donnelly E T , Jacinto M , Liggett S B
Published: July 2002

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.
Analysis of differential gene expression by display of 3' end restriction fragments of cDNAs.
Authors: Prashar Y , Weissman S M
Published: July 2002

We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.
Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.
Authors: Li K J , Garoff H
Published: July 2002

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.
Measuring science: An exploration
Authors: Adams James , Griliches Zvi
Published: July 2002

This paper examines the available United States data on academic research and development (R&D) expenditures and the number of papers published and the number of citations to these papers as possible measures of “output” of this enterprise. We look at these numbers for science and engineering as a whole, for five selected major fields, and at the individual university field level. The published data inScience and Engineering Indicatorsimply sharply diminishing returns to academic R&D using published papers as an “output” measure. These data are quite problematic. Using a newer set of data on papers and citations, based on an “expanding” set of journals and the newly released Bureau of Economic Analysis R&D deflators, changes the picture drastically, eliminating the appearance of diminishing returns but raising the question of why the input prices of academic R&D are rising so much faster than either the gross domestic product deflator or the implicit R&D deflator in industry. A production function analysis of such data at the individual field level follows. It indicates significant diminishing returns to “own” R&D, with the R&D coefficients hovering around 0.5 for estimates with paper numbers as the dependent variable and around 0.6 if total citations are used as the dependent variable. When we substitute scientists and engineers in place of R&D as the right-hand side variables, the coefficient on papers rises from 0.5 to 0.8, and the coefficient on citations rises from 0.6 to 0.9, indicating systematic measurement problems with R&D as the sole input into the production of scientific output. But allowing for individual university field effects drives these numbers down significantly below unity. Because in the aggregate both paper numbers and citations are growing as fast or faster than R&D, this finding can be interpreted as leaving a major, yet unmeasured, role for the contribution of spillovers from other fields, other universities, and other countries.
Different response toPlasmodium falciparummalaria in West African sympatric ethnic groups
Authors: Modiano D. , Petrarca V. , Sirima B. S. , Nebié I. , Diallo D. , Esposito F. , Coluzzi M.
Published: July 2002

The comparison of malaria indicators among populations that have different genetic backgrounds and are uniformly exposed to the same parasite strains is one approach to the study of human heterogeneities in the response to the infection. We report the results of comparative surveys on three sympatric West African ethnic groups, Fulani, Mossi, and Rimaibé, living in the same conditions of hyperendemic transmission in a Sudan savanna area northeast of Ouagadougou, Burkina Faso. The Mossi and Rimaibé are Sudanese negroid populations with a long tradition of sedentary farming, while the Fulani are nomadic pastoralists, partly settled and characterized by non-negroid features of possible caucasoid origin. Parasitological, clinical, and immunological investigations showed consistent interethnic differences inPlasmodium falciparuminfection rates, malaria morbidity, and prevalence and levels of antibodies to variousP. falciparumantigens. The data point to a remarkably similar response to malaria in the Mossi and Rimaibé, while the Fulani are clearly less parasitized, less affected by the disease, and more responsive to all antigens tested. No difference in the use of malaria protective measures was demonstrated that could account for these findings, and sociocultural or environmental factors do not seem to be involved. Known genetic factors of resistance to malaria did not show higher frequencies in the Fulani. The differences in the immune response were not explained by the entomological observations, which indicated substantially uniform exposure to infective bites. The available data support the existence of unknown genetic factors, possibly related to humoral immune responses, determining interethnic differences in the susceptibility to malaria.
A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis.
Authors: Howe J A , Newport J W
Published: July 2002

We have analyzed cyclin E1, a protein that is essential for the G1/S transition, during early development in Xenopus embryos. Cyclin E1 was found to be abundant in eggs, and after fertilization, until the midblastula transition (MBT) when levels of cyclin E1 protein, and associated kinase activity, were found to decline precipitously. Our results suggest that the reduced level of the cyclin E1 protein detected after the MBT does not occur indirectly as a result of degradation of the maternally encoded cyclin E1 mRNA. Instead, the stability of cyclin E1 protein appears to play a major role in reduction of cyclin E1 levels at this time. Cyclin E1 protein was found to be stable during the cleavage divisions but degraded with a much shorter half-life after the MBT. Activation of cyclin E1 protein turnover occurs independent of cell cycle progression, does not require ongoing protein synthesis, and is not triggered as a result of the ratio of nuclei to cytoplasm in embryonic cells that initiates the MBT. We therefore propose that a developmental timing mechanism measures an approximately 5-hr time period, from the time of fertilization, and then allows activation of a protein degradative pathway that regulates cyclin E1. Characterization of the timer suggests that it might be held inactive in eggs by a mitogen-activated protein kinase signal transduction pathway.
Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).
Authors: Fujimoto J , Shiota M , Iwahara T , Seki N , Satoh H , Mori S , Yamamoto T
Published: July 2002

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.
Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to theIdd5diabetes susceptibility region
Authors: Colucci Francesco , Bergman Marie-Louise , Penha-Gonçalves Carlos , Cilio Corrado M. , Holmberg Dan
Published: July 2002

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated fromCtla4andCd28and in fact marks a 20-centimorgan region encompassingIdd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.
Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein
Authors: Li Zhufang , Golub Efim I. , Gupta Ravindra , Radding Charles M.
Published: July 2002

Meiosis-specific homologs of RecA protein have been identified in
Saccharomyces cerevisiae
and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in
Escherichia coli
from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated
k
cat
of 1.5 min
−1
. DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPγS.
A dichotomous role for nitric oxide during acuteToxoplasma gondiiinfection in mice
Authors: Khan Imtiaz A. , Schwartzman Joseph D. , Matsuura Tadashi , Kasper Lloyd H.
Published: July 2002

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, includingToxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally withT. gondiitissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS−/− mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS−/− mice where these tissues appeared normal. Serum interferon γ and tumor necrosis factor α levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection withT. gondiican kill intracellular parasites but can be detrimental, even lethal, to the host.
Transient up-regulation of P2Y2nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes
Authors: Koshiba M. , Apasov S. , Sverdlov V. , Chen P. , Erb L. , Turner J. T. , Weisman G. A. , Sitkovsky M. V.
Published: July 2002

In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T lymphocytes, we observed a transient and protein synthesis-independent enhancement of mRNA expression for the G protein-coupled P2Y2receptor in mouse thymocytes after the addition of steroid hormone or T cell receptor (TCR) crosslinking by anti-TCR mAb. Conversely, dexamethasone-induced increases in mRNA expression for the ligand-gated ion channel P2X1receptor was detected in rat, but not mouse, thymocytes, raising questions about the previously suggested role of P2X1receptors in thymocyte apoptosis. Flow cytometry analysis of thymocyte subsets excluded the possibility that the observed increases in P2Y2receptor mRNA expression were due to the enrichment of steroid-treated cells with an P2Y2mRNA-rich thymocyte subset. Triggering of TCR-mediated intracellular signaling pathways through crosslinking of TCR or by addition of phorbol ester and Ca2+ionophore also resulted in the up-regulation of P2Y2, but not P2X1, receptor mRNA. It is proposed that the rapid increase of P2Y2receptor mRNA expression could be a common early event in responses of T cells to different activating stimuli. Taken together with the recently discovered ability of nucleotide receptor-initiated signaling to antagonize or enhance the effects of TCR crosslinking or steroids on thymocytes, the observed rapid up-regulation of P2Y2receptor mRNA expression may reflect an immediate early gene response where newly expressed cell surface nucleotide receptors provide regulatory feedback signaling from extracellular ATP in the T cell differentiation process.
Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism
Authors: Pontius Brian W. , Lott William B. , von Hippel Peter H.
Published: July 2002

Significant cleavage by hammerhead ribozymes requires activation by
divalent metal ions. Several models have been proposed to account for
the influence of metal ions on hammerhead activity. A number of recent
papers have presented data that have been interpreted as supporting a
one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium
isotope effect has been taken as evidence against a proton transfer in
the rate-limiting step of the cleavage reaction. We propose that these
data are more easily explained by a two-metal-ion mechanism that does
not involve a metal hydroxide, but does involve a proton transfer in
the rate-limiting step.
The origin of red algae: Implications for plastid evolution
Authors: Stiller John W. , Hall Benjamin D.
Published: July 2002

The origin of the red algae has remained an enigma. Historically the Rhodophyta were classified first as plants and later as the most ancient eukaryotic organisms. Recent molecular studies have indicated similarities between red and green plastids, which suggest that there was a single endosymbiotic origin for these organelles in a common ancestor of the rhodophytes and green plants. Previous efforts to confirm or reject this effort by analyses of nuclear DNA have been inconclusive; thus, additional molecular markers are needed to establish the relationship between the host cell lineages, independent of the evolutionary history of their plastids. To furnish such a data set we have sequenced the largest subunit of RNA polymerase II from two red algae, a green alga and a relatively derived amoeboid protist. Phylogenetic analyses provide strong statistical support for an early evolutionary emergence of the Rhodophyta that preceded the origin of the line that led to plants, animals, and fungi. These data, which are congruent with results from extensive analyses of nuclear rDNA, argue for a reexamination of current models of plastid evolution.
Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2
Authors: Ortega Sagrario , Ittmann Michael , Tsang Stephen H. , Ehrlich Michelle , Basilico Claudio
Published: July 2002

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2
in vivo
, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2
−/−
mice are viable, fertile and phenotypically indistinguishable from FGF2
+/+
littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2
−/−
mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.
Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-X
L
and caspase-3-like proteases
Authors: Nagane Motoo , Levitzki Alexander , Gazit Aviv , Cavenee Webster K. , Huang H.-J. Su
Published: July 2002

Alterations of the epidermal growth factor receptor (
EGFR
) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2–7, resulting in truncation of the extracellular domain (ΔEGFR or EGFRvIII), which occurs in a large fraction of
de novo
malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-X
L
. Here, we demonstrate that the ΔEGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-X
L
was elevated in U87MG.ΔEGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.ΔEGFR cells. These responses were highly specific to constitutively kinase-active ΔEGFR, because overexpression of kinase-deficient ΔEGFR (DK) or wild-type EGFR had no such effects. Correspondingly, ΔEGFR specific tyrosine kinase inhibitors reduced Bcl-X
L
expression and potentiated CDDP-induced apoptosis in U87MG.ΔEGFR cells. Ectopic overexpression of Bcl-X
L
in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing ΔEGFR.
A release mechanism for stored ATP in ocular ciliary epithelial cells
Authors: Mitchell Claire H. , Carré David A. , McGlinn Alice M. , Stone Richard A. , Civan Mortimer M.
Published: July 2002

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl
−
-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.
LCK-phosphorylated human killer cell-inhibitory receptors recruit and activate phosphatidylinositol 3-kinase
Authors: Marti Francesc , Xu C. Wilson , Selvakumar Annamalai , Brent Roger , Dupont Bo , King Philip D.
Published: July 2002

HLA-specific killer cell inhibitory receptors (KIR) are thought to impede natural killer (NK) and T cell activation programs through recruitment of the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, to their cytoplasmic tails (CYT). To identify other SH2 domain-containing proteins that bind KIR CYT, we used the recently described yeast two-bait interaction trap and a modified version of this system, both of which permit tyrosine phosphorylation of bait proteins. Using these systems, we show that KIR CYT, once phosphorylated by the src-family tyrosine kinase LCK, additionally bind the p85α regulatory subunit of phosphatidylinositol (PI) 3-kinase. Furthermore, we show that in an NK cell line, NK3.3, cross-linking of KIR results in recruitment of p85α to KIR and activation of PI 3-kinase lipid kinase activity. One consequence of KIR coupling to PI 3-kinase is downstream activation of the antiapoptotic protein kinase AKT. Therefore, in addition to providing negative signals, KIR may also contribute positive signals for NK and T cell growth and/or survival.
Rap1 protein regulates telomere turnover in yeast
Authors: Krauskopf Anat , Blackburn Elizabeth H.
Published: July 2002

Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein–DNA complex. Previous work has provided evidence that in the budding yeasts
Kluyveromyces lactis
and
Saccharomyces cerevisiae
, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in
K. lactis,
truncating the Rap1p C-terminal tail (Rap1p-ΔC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-ΔC mutation with a telomerase template mutation (
ter1-kpn
), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats.
δ-Tonoplast intrinsic protein defines unique plant vacuole functions
Authors: Jauh Guang-Yuh , Fischer Andreas M. , Grimes Howard D. , Ryan Clarence A. , Rogers John C.
Published: July 2002

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), δ-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of δ-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.
The von Hippel–Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal
Authors: Pause Arnim , Lee Stephen , Lonergan Kim M. , Klausner Richard D.
Published: July 2002

The inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene predisposes affected individuals to the human VHL cancer syndrome and is associated with sporadic renal cell carcinomas (RCC) and brain hemangioblastomas. VHL-negative 786–0 RCC cells are tumorigenic in nude mice which is suppressed by the reintroduction of VHL. Remarkably, this occurs without affecting the growth rate and cell cycle profile of these cells in culture. The 786–0 cell line, like many cancer cells, fails to exit the cell cycle upon serum withdrawal. Here, it is shown that reintroduction of the wild-type VHL gene restores the ability of VHL-negative RCC cancer cells to exit the cell cycle and enter G
0
/quiescence in low serum. Both VHL-positive and VHL-negative RCC cells exit the cell cycle by contact inhibition. The cyclin-dependent kinase inhibitor, p27, accumulates upon serum withdrawal, only in the presence of VHL, as a result of the stabilization of the protein. We propose that the loss of wild-type VHL gene results in a specific cellular defect in serum-dependent growth control, which may initiate tumor formation. This is corrected by the reintroduction of wild-type VHL, implicating VHL as the first tumor suppressor involved in the regulation of cell cycle exit, which is consistent with its gatekeeper function in the kidney.
Mirasorvone: A masked 20-ketopregnane from the defensive secretion of a diving beetle (
Thermonectus marmoratus
)
Authors: Meinwald Jerrold , Huang Qing , Vrkoč Jan , Herath Kithsiri B. , Yang Zhi-Cai , Schröder Frank , Attygalle Athula B. , Iyengar Vikram K. , Morgan Randy C. , Eisner Thomas
Published: July 2002

The sunburst diving beetle,
Thermonectus marmoratus
, ejects a milky fluid from its prothoracic defensive glands when disturbed. Two major volatile components of this secretion are steroids; cybisterone (structure 7) constitutes about 20% of the volatiles, and a new steroid, mirasorvone, about 50%. Mirasorvone is assigned an 18-oxygenated pregnane structure (structure 9) on the basis of extensive spectroscopic data. Although no 18-oxygenated steroid has been described previously from an insect source, a closely related hormone with mineralocorticoid activity, 18-hydroxydeoxycorticosterone (structure 13), has been isolated from the adrenal glands of rats.
Multiple neurological abnormalities in mice deficient in the G protein G
o
Authors: Jiang Meisheng , Gold Michael S. , Boulay Guylain , Spicher Karsten , Peyton Michael , Brabet Philippe , Srinivasan Yogambul , Rudolph Uwe , Ellison Gaylord , Birnbaumer Lutz
Published: July 2002

The G protein G
o
is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, α
2
-adrenergic, M2 muscarinic, and somatostatin receptors.
In vitro
, G
o
is also activated by growth cone-associated protein of
M
r
43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that G
o
may play in intact cells and whole body homeostasis, we disrupted the gene encoding the α subunits of G
o
in embryonic stem cells and derived G
o
-deficient mice. Mice with a disrupted α
o
gene (α
o
−/− mice) lived but had an average half-life of only about 7 weeks. No G
o
α was detectable in homogenates of α
o
−/− mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50–55% at saturation) was unaffected, but inhibition of Ca
2+
channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the α
o
−/− cells examined, the Ca
2+
channel was activated at voltages that were 13.3 ± 1.7 mV lower than in their counterparts. Loss of α
o
was not accompanied by appearance of significant amounts of active free βγ dimers (prepulse test). At the level of the living animal, G
o
-deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, α
o
−/− mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all α
o
−/− mice turned only counterclockwise. These findings indicate that G
o
plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of G
o
in Ca
2+
channel regulation by an unknown mechanism.
α
2
-macroglobulin associates with β-amyloid peptide and prevents fibril formation
Authors: Hughes Stephen R. , Khorkova Olga , Goyal Shefali , Knaeblein Joerg , Heroux Jeffrey , Riedel Norbert G. , Sahasrabudhe Sudhir
Published: July 2002

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa β-amyloid peptide (Aβ), a major constituent of senile plaques in Alzheimer’s disease. The carboxy terminus of α
2
-macroglobulin (α2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Aβ, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of α2M with Aβ from the yeast cell, and by formation of SDS-resistant Aβ complexes in polyacrylamide gels by using synthetic Aβ and purified α2M. The association of Aβ with α2M and various purified amyloid binding proteins was assessed by employing a method measuring protein–protein interactions in liquid phase. The dissociation constant by this technique for the α2M–Aβ association using labeled purified proteins was measured (
K
d
= 350 nM). Electron microscopy showed that a 1:8 ratio of α2M to Aβ prevented fibril formation in solution; the same ratio to Aβ of another acute phase protein, α
1
-antichymotrypsin, was not active in preventing fibril formation
in vitro
. These results were corroborated by data obtained from an
in vitro
aggregation assay employing Thioflavine T. The interaction of α2M with Aβ suggests new pathway(s) for the clearance of the soluble amyloid peptide.
Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity
Authors: Mandelboim Ofer , Malik Pratap , Davis Daniel M. , Jo Chang H. , Boyson Jonathan E. , Strominger Jack L.
Published: July 2002

In addition to their role in peptide antigen presentation, class I MHC proteins also play a critical role in inhibiting natural killer (NK) cytotoxicity through interaction with NK inhibitory receptors. Thus, NK cells are cytotoxic to virus-infected and tumor cells that have lost class I MHC protein expression. However, the nature of the receptors involved in the triggering of lysis of target cells is poorly understood. CD16 (Fcγ receptor III) has been described as a receptor expressed on NK cells that facilitates antibody-dependent cellular cytotoxicity (ADCC) by binding to the Fc portion of various antibodies. However, we show here that CD16 has a broader function and is directly involved in the lysis of some virus-infected cells and tumor cells, independent of antibody binding. The presence of a putative CD16 ligand on appropriate target cells has also been demonstrated by the use of a CD16-Ig fusion protein.
Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication
Authors: Flores Osvaldo , Lee Gary , Kessler Joseph , Miller Michael , Schlief William , Tomassini Joanne , Hazuda Daria
Published: July 2002

HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.
Somatic stiffness of cochlear outer hair cells is voltage-dependent
Authors: He David Z. Z. , Dallos Peter
Published: July 2002

The mammalian cochlea depends on an amplification process for its sensitivity and frequency-resolving capability. Outer hair cells are responsible for providing this amplification. It is usually assumed that the membrane–potential-driven somatic shape changes of these cells are the basis of the amplifying process. It is of interest to see whether mechanical reactance changes of the cells might accompany their changes in cell shape. We now show that the cylindrical outer hair cells change their axial stiffness as their membrane potential is altered. Cell stiffness was determined by optoelectronically measuring the amplitude of motion of a flexible vibrating fiber as it was loaded by the isolated cell. Voltage commands to the cell were delivered in a tight–seal whole–cell configuration. Cell stiffness was decreased by depolarization and increased by hyperpolarization.
Medin: An integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid
Authors: Häggqvist Bo , Näslund Jan , Sletten Knut , Westermark Gunilla T. , Mucchiano Gerd , Tjernberg Lars O. , Nordstedt Christer , Engström Ulla , Westermark Per
Published: July 2002

Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. The importance and impact of the amyloid deposits are unknown. In this study we have purified a 5.5-kDa aortic medial amyloid component, by size-exclusion chromatography and RP-HPLC, from three individuals, and we have shown by amino acid sequence analysis that the amyloid is derived from an integral proteolytic fragment of lactadherin. Lactadherin is a 364-aa glycoprotein, previously known to be expressed by mammary epithelial cells as a cell surface protein and secreted as part of the milk fat globule membrane. The multidomain protein has a C-terminal domain showing homology to blood coagulation factors V and VIII. We found that the main constituent of aortic medial amyloid is a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By using
in situ
hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic peptides forms amyloid-like fibrils
in vitro
. Lactadherin was not previously known to be an amyloid precursor protein or to be expressed in aortic tissue. The structure of lactadherin may implicate an important regulatory function in the aorta.
Targeted manipulation of maize genes
in vivo
using chimeric RNA/DNA oligonucleotides
Authors: Zhu Tong , Peterson David J. , Tagliani Laura , St. Clair Grace , Baszczynski Chris L. , Bowen Ben
Published: July 2002

Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA/DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green fluorescence protein transgene was site-specifically modified
in vivo
. Expression of the introduced inactive green fluorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10
−4
, which is 1–3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.
N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA
2
subunit to form an N cap that terminates the triple-stranded coiled coil
Authors: Chen Jue , Skehel John J. , Wiley Don C.
Published: July 2002

The structure of a stable recombinant ectodomain of influenza hemagglutinin HA
2
subunit, EHA
2
(23–185), defined by proteolysis studies of the intact bacterial-expressed ectodomain, was determined to 1.9-Å resolution by using x-ray crystallography. The structure reveals a domain composed of N- and C-terminal residues that form an N cap terminating both the N-terminal α-helix and the central coiled coil. The N cap is formed by a conserved sequence, and part of it is found in the neutral pH conformation of HA. The C-terminal 23 residues of the ectodomain form a 72-Å long nonhelical structure ordered to within 7 residues of the transmembrane anchor. The structure implies that continuous α helices are not required for membrane fusion at either the N or C termini. The difference in stability between recombinant molecules with and without the N cap sequences suggests that additional free energy for membrane fusion may become available after the formation of the central triple-stranded coiled coil and insertion of the fusion peptide into the target membrane.
Contextual influences in V1 as a basis for pop out and asymmetry in visual search
Authors: Li Zhaoping
Published: July 2002

I use a model to show how simple, bottom-up, neural mechanisms in primary visual cortex can qualitatively explain the preattentive component of complex psychophysical phenomena of visual search for a target among distracters. Depending on the image features, the speed of search ranges from fast, when a target pops-out or is instantaneously detectable, to very slow, and it can be asymmetric with respect to switches between the target and distracter objects. It has been unclear which neural mechanisms or even cortical areas control the ease of search, and no physiological correlate has been found for search asymmetry. My model suggests that contextual influences in V1 play a significant role.
Frontiers of finance: Evolution and efficient markets
Authors: Farmer J. Doyne , Lo Andrew W.
Published: July 2002

In this review article, we explore several recent advances in
the quantitative modeling of financial markets. We begin with the
Efficient Markets Hypothesis and describe how this controversial idea
has stimulated a number of new directions of research, some focusing on
more elaborate mathematical models that are capable of rationalizing
the empirical facts, others taking a completely different tack in
rejecting rationality altogether. One of the most promising directions
is to view financial markets from a biological perspective and,
specifically, within an evolutionary framework in which markets,
instruments, institutions, and investors interact and evolve
dynamically according to the “law” of economic selection. Under
this view, financial agents compete and adapt, but they do not
necessarily do so in an optimal fashion. Evolutionary and ecological
models of financial markets is truly a new frontier whose exploration
has just begun.
Pollinator preference and the evolution of floral traits in monkeyflowers (
Mimulus
)
Authors: Schemske Douglas W. , Bradshaw H. D.
Published: July 2002

A paradigm of evolutionary biology is that adaptation and reproductive isolation are caused by a nearly infinite number of mutations of individually small effect. Here, we test this hypothesis by investigating the genetic basis of pollinator discrimination in two closely related species of monkeyflowers that differ in their major pollinators. This system provides a unique opportunity to investigate the genetic architecture of adaptation and speciation because floral traits that confer pollinator specificity also contribute to premating reproductive isolation. We asked: (
i
) What floral traits cause pollinator discrimination among plant species? and (
ii
) What is the genetic basis of these traits? We examined these questions by using data obtained from a large-scale field experiment where genetic markers were employed to determine the genetic basis of pollinator visitation. Observations of F
2
hybrids produced by crossing bee-pollinated
Mimulus lewisii
with hummingbird-pollinated
Mimulus cardinalis
revealed that bees preferred large flowers low in anthocyanin and carotenoid pigments, whereas hummingbirds favored nectar-rich flowers high in anthocyanins. An allele that increases petal carotenoid concentration reduced bee visitation by 80%, whereas an allele that increases nectar production doubled hummingbird visitation. These results suggest that genes of large effect on pollinator preference have contributed to floral evolution and premating reproductive isolation in these monkeyflowers. This work contributes to growing evidence that adaptation and reproductive isolation may often involve major genes.
DNA transport by a micromachined Brownian ratchet device
Authors: Bader Joel S. , Hammond Richard W. , Henck Steven A. , Deem Michael W. , McDermott Gregory A. , Bustillo James M. , Simpson John W. , Mulhern Gregory T. , Rothberg Jonathan M.
Published: July 2002

We have micromachined a silicon-chip device that transports DNA with a Brownian ratchet that rectifies the Brownian motion of microscopic particles. Transport properties for a DNA 50-mer agree with theoretical predictions, and the DNA diffusion constant agrees with previous experiments. This type of micromachine could provide a generic pump or separation component for DNA or other charged species as part of a microscale lab-on-a-chip. A device with reduced feature size could produce a size-based separation of DNA molecules, with applications including the detection of single-nucleotide polymorphisms.