bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis
Authors: Borsig Lubor , Wong Richard , Hynes Richard O. , Varki Nissi M. , Varki Ajit
Published: July 2002

P-selectin facilitates human carcinoma metastasis in immunodeficient mice by mediating early interactions of platelets with bloodborne tumor cells via their cell surface mucins, and this process can be blocked by heparin [Borsig, L., Wong, R., Feramisco, J., Nadeau, D. R., Varki, N. M. & Varki, A. (2001)Proc. Natl. Acad. Sci. USA98, 3352–3357]. Here we show similar findings with a murine adenocarcinoma in syngeneic immunocompetent mice but involving a different P-selectin ligand, possibly a sulfated glycolipid. Thus, metastatic spread can be facilitated by tumor cell selectin ligands other than mucins. Surprisingly, L-selectin expressed on endogenous leukocytes also facilitates metastasis in both the syngeneic and xenogeneic (T and B lymphocyte deficient) systems. PL-selectin double deficient mice show that the two selectins work synergistically. Although heparin can block both P- and L-selectinin vitro, thein vivoeffect of a single heparin dose given before tumor cells seems to be completely accounted for by blockade of P-selectin function. Thus, L-selectin on neutrophils, monocytes, and/or NK cells has a role in facilitating metastasis, acting beyond the early time points wherein P-selectin mediates interactions of platelet with tumor cells.
Angiotensin II type 2 receptor mediates programmed cell death.
Authors: Yamada T , Horiuchi M , Dzau V J
Published: July 2002

The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase). Vanadate, a protein-tyrosine-phosphatase inhibitor, attenuated the dephosphorylation of MAP kinases by the AT2 receptor and restored the apoptotic changes. Antisense oligonucleotide to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor-mediated apoptosis. These results suggest that protein-tyrosine-phosphatase, including MAP kinase phosphatase 1 activated by the AT2 receptor, is involved in apoptosis. We hypothesize that this apoptotic function of the AT2 receptor may play an important role in developmental biology and pathophysiology.
Responses of the phototransduction cascade to dim light.
Authors: Langlois G , Chen C K , Palczewski K , Hurley J B , Vuong T M
Published: July 2002

The biochemistry of visual excitation is kinetically explored by measuring the activity of the cGMP phosphodiesterase (PDE) at light levels that activate only a few tens of rhodopsin molecules per rod. At 23 degrees C and in the presence of ATP, the pulse of PDE activity lasts 4 s (full width at half maximum). Complementing the rod outer segments (ROS) with rhodopsin kinase (RK) and arrestin or its splice variant p44 does not significantly shorten the pulse. But when the ROS are washed, the duration of the signal doubles. Adding either arrestin or p44 back to washed ROS approximately restores the pulse width to its initial value, with p44 being 10 times more efficient than arrestin. This supports the idea that, in vivo, capping of phosphorylated R* is mostly done by p44. When myristoylated (14:0) recoverin is added to unwashed ROS, the pulse duration and amplitude increase by about 50% if the free calcium is 500 nM. This effect increases further if the calcium is raised to 1 microM. Whenever R* deactivation is changed--when RK is exogenously enriched or when ATP is omitted from the buffer--there is no impact on the rising slope of the PDE pulse but only on its amplitude and duration. We explain this effect as due to the unequal competition between transducin and RK for R*. The kinetic model issued from this idea fits the data well, and its prediction that enrichment with transducin should lengthen the PDE pulse is successfully validated.
Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.
Authors: Said S I , Berisha H I , Pakbaz H
Published: July 2002

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.
Behavioral stress modifies hippocampal plasticity through N-methyl-D-aspartate receptor activation.
Authors: Kim J J , Foy M R , Thompson R F
Published: July 2002

Behavioral stress has detrimental effects on subsequent cognitive performance in many species, including humans. For example, humans exposed to stressful situations typically exhibit marked deficits in various learning and memory tasks. However, the underlying neural mechanisms by which stress exerts its effects on learning and memory are unknown. We now report that in adult male rats, stress (i.e., restraint plus tailshock) impairs long-term potentiation (LTP) but enhances long-term depression (LTD) in the CA1 area of the hippocampus, a structure implicated in learning and memory processes. These effects on LTP and LTD are prevented when the animals were given CGP39551 (the carboxyethylester of CGP 37849; DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, before experiencing stress. In contrast, the anxiolytic drug diazepam did not block the stress effects on hippocampal plasticity. Thus, the effects of stress on subsequent LTP and LTD appear to be mediated through the activation of the NMDA subtype of glutamate receptors. Such modifications in hippocampal plasticity may contribute to learning and memory impairments associated with stress.
Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.
Authors: Wolff S , Afzal V
Published: July 2002

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.
Calcium-dependent oligonucleotide antagonists specific for L-selectin.
Authors: O'Connell D , Koenig A , Jennings S , Hicke B , Han H L , Fitzwater T , Chang Y F , Varki N , Parma D , Varki A
Published: July 2002

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.
Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.
Authors: McCall M A , Gregg R G , Behringer R R , Brenner M , Delaney C L , Galbreath E J , Zhang C L , Pearce R A , Chiu S Y , Messing A
Published: July 2002

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.
A new extinct primate among the Pleistocene megafauna of Bahia, Brazil.
Authors: Cartelle C , Hartwig W C
Published: July 2002

A nearly complete skeleton of a robust-bodied New World monkey that resembles living spider monkeys was recovered from undisturbed Pleistocene deposits in the Brazilian state of Bahia. The skeleton displays the highly specialized postcranial pattern typical of spider and woolly spider monkeys and shares cranial similarities to the spider monkey exclusively. It is generically distinct on the basis of its robustness (>20 kg) and on the shape of its braincase. This new genus indicates that New World monkeys nearly twice the size of those living today were part of the mammalian biomass of southern Amazonia in the late Pleistocene. The discovery of this specimen expands the known adaptive diversity of New World monkeys and demonstrates that they underwent body size expansion in the terminal Pleistocene, as did many other types of mammals.
Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.
Authors: Smit M J , Leurs R , Alewijnse A E , Blauw J , Van Nieuw Amerongen G P , Van De Vrede Y , Roovers E , Timmerman H
Published: July 2002

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.
Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.
Authors: Sugimoto K , Shimomura T , Hashimoto K , Araki H , Sugino A , Matsumoto K
Published: July 2002

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.
SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.
Authors: Chen J D , Umesono K , Evans R M
Published: July 2002

Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling.
Interaction of the protein import and folding machineries of the chloroplast.
Authors: Kessler F , Blobel G
Published: July 2002

We report the molecular cloning of import intermediate associated protein (IAP) 100, a 100-kDa protein of the chloroplast protein import machinery of peas. IAP100 contains two potential alpha-helical transmembrane segments and also behaves like an integral membrane protein. It was localized to the inner chloroplast envelope membrane. Immunoprecipitation experiments using monospecific anti-IAP100 antibodies and a nonionic detergent-generated chloroplast lysate gave the following results. (i) The four integral membrane proteins of the outer chloroplast import machinery were not coprecipitated with IAP100 indicating that the inner and outer membrane import machineries are not coupled in isolated chloroplasts. (ii) the major protein that coprecipitated with IAP100 was identified as stromal chaperonin 60 (cpn60); the association of IAP100 and cpn60 was specific and was abolished when immunoprecipitation was carried out in the presence of ATP. (iii) In a lysate from chloroplasts that had been preincubated for various lengths of time in an import reaction with radiolabeled precursor (pS) of the small subunit of Rubisco, we detected coimmunoprecipitation of IAP100, cpn60, and the imported mature form (S) of precursor. Relative to the time course of import, coprecipitation of S first increased and then decreased, consistent with a transient association of the newly imported S with the chaperonin bound to IAP100. These data suggest that IAP100 serves in recruiting chaperonin for folding of newly imported proteins.
Amygdala activity at encoding correlated with long-term, free recall of emotional information.
Authors: Cahill L , Haier R J , Fallon J , Alkire M T , Tang C , Keator D , Wu J , McGaugh J L
Published: July 2002

Positron emission tomography of cerebral glucose metabolism in adult human subjects was used to investigate amygdaloid complex (AC) activity associated with the storage of long-term memory for emotionally arousing events. Subjects viewed two videos (one in each of two separate positron emission tomography sessions, separated by 3-7 days) consisting either of 12 emotionally arousing film clips ("E" film session) or of 12 relatively emotionally neutral film clips ("N" film session), and rated their emotional reaction to each film clip immediately after viewing it. Three weeks after the second session, memory for the videos was assessed in a free recall test. As expected, the subjects' average emotional reaction to the E films was higher than that for the N films. In addition, the subjects recalled significantly more E films than N films. Glucose metabolic rate of the right AC while viewing the E films was highly correlated with the number of E films recalled. AC activity was not significantly correlated with the number of N films recalled. The findings support the view derived from both animal and human investigations that the AC is selectively involved with the formation of enhanced long-term memory associated with emotionally arousing events.
A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.
Authors: Bureau T E , Ronald P C , Wessler S R
Published: July 2002

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.
SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.
Authors: Jacobsen S E , Binkowski K A , Olszewski N E
Published: July 2002

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.
Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.
Authors: Santoni-Rugiu E , Preisegger K H , Kiss A , Audolfsson T , Shiota G , Schmidt E V , Thorgeirsson S S
Published: July 2002

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.
Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.
Authors: Wong M H , Hermiston M L , Syder A J , Gordon J I
Published: July 2002

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.
Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance.
Authors: Buckle M , Williams R M , Negroni M , Buc H
Published: July 2002

A rapid direct assay for polymerase-induced elongation along a given template is an obligate requirement for understanding the processivity of polymerization and the mode of action of drugs and inhibitors on this process. Surface plasmon resonance can be used to follow the association and the dissociation rates of a given reverse transcriptase on DNA.RNA and DNA.DNA hybrids immobilized on a biotin-streptavidin surface. The addition of nucleotides complementary to the template strand produces an increase in the local mass, as deduced from an increase in the measured signal, due to elongation of the primer strand that allows an estimation of both the extent and rate of the polymerization process. The terminator drug 3'-deoxy-3'-azidothymidine triphosphate completely abolishes the increase in signal as would be expected from an inhibition of elongation. This technique provides a sensitive assay for the affinities of different polymerases for specific templates and for the effects of terminators of the elongation process.
The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.
Authors: Gottardi C J , Arpin M , Fanning A S , Louvard D
Published: July 2002

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.
DNA repeats identify novel virulence genes in Haemophilus influenzae.
Authors: Hood D W , Deadman M E , Jennings M P , Bisercic M , Fleischmann R D , Venter J C , Moxon E R
Published: July 2002

The whole genome sequence (1.83 Mbp) of Haemophilus influenzae strain Rd was searched to identify tandem oligonucleotide repeat sequences. Loss or gain of one or more nucleotide repeats through a recombination-independent slippage mechanism is known to mediate phase variation of surface molecules of pathogenic bacteria, including H. influenzae. This facilitates evasion of host defenses and adaptation to the varying microenvironments of the host. We reasoned that iterative nucleotides could identify novel genes relevant to microbe-host interactions. Our search of the Rd genome sequence identified 9 novel loci with multiple (range 6-36, mean 22) tandem tetranucleotide repeats. All were found to be located within putative open reading frames and included homologues of hemoglobin-binding proteins of Neisseria, a glycosyltransferase (IgtC gene product) of Neisseria, and an adhesin of Yersinia. These tetranucleotide repeat sequences were also shown to be present in two other epidemiologically different H. influenzae type b strains, although the number and distribution of repeats was different. Further characterization of the IgtC gene showed that it was involved in phenotypic switching of a lipopolysaccharide epitope and that this variable expression was associated with changes in the number of tetranucleotide repeats. Mutation of IgtC resulted in attenuated virulence of H. influenzae in an infant rat model of invasive infection. These data indicate the rapidity, economy, and completeness with which whole genome sequences can be used to investigate the biology of pathogenic bacteria.
Efficiency of DNA replication in the polymerase chain reaction
Authors: Stolovitzky Gustavo , Cecchi Guillermo
Published: July 2002

A detailed quantitative kinetic model for the polymerase chain
reaction (PCR) is developed, which allows us to predict the probability
of replication of a DNA molecule in terms of the physical parameters
involved in the system. The important issue of the determination of the
number of PCR cycles during which this probability can be considered to
be a constant is solved within the framework of the model. New
phenomena of multimodality and scaling behavior in the distribution of
the number of molecules after a given number of PCR cycles are
presented. The relevance of the model for quantitative PCR is
discussed, and a novel quantitative PCR technique is proposed.
Neural mechanisms for visual memory and their role in attention
Authors: Desimone Robert
Published: July 2002

Recent studies show that neuronal mechanisms for learning and memory both dynamically modulate and permanently alter the representations of visual stimuli in the adult monkey cortex. Three commonly observed neuronal effects in memory-demanding tasks are repetition suppression, enhancement, and delay activity. In repetition suppression, repeated experience with the same visual stimulus leads to both short- and long-term suppression of neuronal responses in subpopulations of visual neurons. Enhancement works in an opposite fashion, in that neuronal responses are enhanced for objects with learned behavioral relevance. Delay activity is found in tasks in which animals are required to actively hold specific information “on-line” for short periods. Repetition suppression appears to be an intrinsic property of visual cortical areas such as inferior temporal cortex and is thought to be important for perceptual learning and priming. By contrast, enhancement and delay activity may depend on feedback to temporal cortex from prefrontal cortex and are thought to be important for working memory. All of these mnemonic effects on neuronal responses bias the competitive interactions that take place between stimulus representations in the cortex when there is more than one stimulus in the visual field. As a result, memory will often determine the winner of these competitions and, thus, will determine which stimulus is attended.
Transgenic mice carrying the dominant rhodopsin mutation P347S: Evidence for defective vectorial transport of rhodopsin to the outer segments
Authors: Li Tiansen , Snyder Wendy K. , Olsson Jane E. , Dryja Thaddeus P.
Published: July 2002

To explore the pathogenic mechanism of dominant mutations affecting
the carboxyl terminus of rhodopsin that cause retinitis pigmentosa, we
generated five lines of transgenic mice carrying the proline-347 to
serine (P347S) mutation. The severity of photoreceptor degeneration
correlated with the levels of transgene expression in these lines.
Visual function as measured by the electroretinogram was approximately
normal at an early age when there was little histologic evidence of
photoreceptor degeneration, but it deteriorated as photoreceptors
degenerated. Immunocytochemical staining showed the mutant rhodopsin
predominantly in the outer segments prior to histologically evident
degeneration, a finding supported by quantitation of signal intensities
in different regions of the photoreceptor cells by confocal microscopy.
A distinct histopathologic abnormality was the accumulation of
submicrometer-sized vesicles extracellularly near the junction between
inner and outer segments. The extracellular vesicles were bound by a
single membrane that apparently contained rhodopsin as revealed by
ultrastructural immunocytochemical staining with anti-rhodopsin
antibodies. The outer segments, although shortened, contained
well-packed discs. Proliferation of the endoplasmic reticulum as
reported in
Drosophila
expressing dominant rhodopsin
mutations was not observed. The accumulation of rhodopsin-laden
vesicles likely represents aberrant transport of rhodopsin from the
inner segments to the nascent disc membranes of the outer segments. It
is possible that photoreceptor degeneration occurs because of a failure
to renew outer segments at a normal rate, thereby leading to a
progressive shortening of outer segments, or because of the loss of
cellular contents to the extracellular space, or because of both.
Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein
Authors: Ramprasad Mysore P. , Terpstra Valeska , Kondratenko Nonna , Quehenberger Oswald , Steinberg Daniel
Published: July 2002

We have previously identified a 94- to 97-kDa oxidized low
density lipoprotein (LDL)-binding protein in mouse macrophages as
macrosialin (MS), a member of the lamp family. Earlier immunostaining
studies have shown that MS and its human homolog, CD68, are
predominantly intracellular proteins. However, using sensitive
techniques such as flow cytometry (FACS) and cell-surface-specific
biotinylation, we now show that there is significant surface expression
of these proteins. FACS analysis of intact cells using mAb FA/11
showed small but definite surface expression of MS in resident mouse
peritoneal macrophages but this was greatly enhanced with
thioglycollate elicitation. Biotinylation of intact cells and
detergent-solubilized cell preparations followed by immunoprecipitation
revealed 10–15% of the total MS content of elicited macrophages on
the plasma membrane. Similar results were obtained with untreated RAW
264.7 cells. FACS analysis of intact THP-1 monocytic cells showed
minimal surface expression of CD68 on unactivated cells (4% of total
cell content). Stimulation with phorbol 12-myristate 13-acetate
increased both surface and total CD68 expression considerably.
Furthermore, the specific binding at 4°C and uptake at 37°C of
125
I-labeled oxidized LDL by activated THP-1 cells was
inhibited by 30–50% by CD68 mAbs KP-1 and EBM-11. Thus, although the
surface expression of MS/CD68 at steady-state represents only a small
percentage of their total cellular content, these proteins can play a
significant role in oxidized LDL uptake by activated macrophages
in vitro
and could contribute to foam cell formation in
atherosclerotic lesions.
Sensitivity to inhibition by β-chemokines correlates with biological phenotypes of primary HIV-1 isolates
Authors: Jansson Marianne , Popovic Mikulas , Karlsson Anders , Cocchi Fiorenza , Rossi Paolo , Albert Jan , Wigzell Hans
Published: July 2002

Primary HIV-1 isolates were evaluated for their sensitivity to inhibition by β-chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Virus isolates of both nonsyncytium-inducing (NSI) and syncytium-inducing (SI) biological phenotypes recovered from patients at various stages of HIV-1 infection were assessed, and the results indicated that only the isolates with the NSI phenotype were substantially inhibited by the β-chemokines. More important to note, these data demonstrate that resistance to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β is not restricted to T cell line-adapted SI isolates but is also a consistent property among primary SI isolates. Analysis of isolates obtained sequentially from infected individuals in whom viruses shifted from NSI to SI phenotype during clinical progression exhibited a parallel loss of sensitivity to β-chemokines. Loss of virus sensitivity to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β was furthermore associated with changes in the third variable (V3) region amino acid residues previously described to correlate with a shift of virus phenotype from NSI to SI. Of interest, an intermediate V3 genotype correlated with a partial inhibition by the β-chemokines. In addition, we also identified viruses sensitive to RANTES, MIP-1α, and MIP-1β of NSI phenotype that were isolated from individuals with AIDS manifestations, indicating that loss of sensitivity to β-chemokine inhibition and shift in viral phenotype are not necessarily prerequisites for the pathogenesis of HIV-1 infection.
On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.
Authors: Lomakin A , Chung D S , Benedek G B , Kirschner D A , Teplow D B
Published: July 2002

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.
In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.
Authors: Borden K L , Lally J M , Martin S R , O'Reilly N J , Solomon E , Freemont P S
Published: July 2002

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.
Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.
Authors: Lambert D M , Barney S , Lambert A L , Guthrie K , Medinas R , Davis D E , Bucy T , Erickson J , Merutka G , Petteway S R
Published: July 2002

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.
The rate constant of photoinhibition, measured in lincomycin-treated leaves, is directly proportional to light intensity.
Authors: Tyystjärvi E , Aro E M
Published: July 2002

Pumpkin leaves grown under high light (500-700 micromol of photons m-2.s-1) were illuminated under photon flux densities ranging from 6.5 to 1500 micromol.m-2.s-1 in the presence of lincomycin, an inhibitor of chloroplast protein synthesis. The illumination at all light intensities caused photoinhibition, measured as a decrease in the ratio of variable to maximum fluorescence. Loss of photosystem II (PSII) electron transfer activity correlated with the decrease in the fluorescence ratio. The rate constant of photoinhibition, determined from first-order fits, was directly proportional to photon flux density at all light intensities studied. The fluorescence ratio did not decrease if the leaves were illuminated in low light in the absence of lincomycin or incubated in darkness in the presence of lincomycin. The constancy of the quantum yield of photoinhibition under different photon flux densities strongly suggests that photoinhibition in vivo occurs by one dominant mechanism under all light intensities. This mechanism probably is not the acceptor side mechanism characterized in the anaerobic case in vitro. Furthermore, there was an excellent correlation between the loss of PSII activity and the loss of the D1 protein from thylakoid membranes under low light. At low light, photoinhibition occurs so slowly that inactive PSII centers with the D1 protein waiting to be degraded do not accumulate. The kinetic agreement between D1 protein degradation and the inactivation of PSII indicates that the turnover of the D1 protein depends on photoinhibition under both low and high light.
A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry.
Authors: Steinman L
Published: July 2002

Inflammatory infiltrates in tissue-specific autoimmune disease comprise a collection of T cells with specificity for an antigen in the target organ. These specific cells recruit a population of nonspecific T cells and macrophages. The rare tissue-specific T cells in the infiltrate have the capacity to regulate both the influx and the efflux of cells from the tissue. Administration of an altered peptide ligand for the specific T cell which triggers autoimmunity can lead to the regression of the entire inflammatory ensemble in a few hours. Interleukin 4 is a critical cytokine involved in the regression of the inflammatory infiltrate.
Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage.
Authors: Park J Y , Shigenaga M K , Ames B N
Published: July 2002

Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.