bluuebunny/crossref_metadata_2025 huggingface dataset:
Beyond superquenching: Hyper-efficient energy transfer from conjugated polymers to gold nanoparticles
Authors: Fan Chunhai , Wang Shu , Hong Janice W. , Bazan Guillermo C. , Plaxco Kevin W. , Heeger Alan J.
Published: May 2003

Gold nanoparticles quench the fluorescence of cationic polyfluorene with Stern–Volmer constants (
K

SV

) approaching 10
11
M
—
1
, several orders of magnitude larger than any previously reported conjugated polymer–quencher pair and 9–10 orders of magnitude larger than small molecule dye–quencher pairs. The dependence of

K
SV

on ionic strength, charge and conjugation length of the polymer, and the dimensions (and thus optical properties) of the nanoparticles suggests that three factors account for this extraordinary efficiency: (
i
) amplification of the quenching via rapid internal energy or electron transfer, (
ii
) electrostatic interactions between the cationic polymer and anionic nanoparticles, and (
iii
) the ability of gold nanoparticles to quench via efficient energy transfer. As a result of this extraordinarily high

K
SV

, quenching can be observed even at subpicomolar concentrations of nanoparticles, suggesting that the combination of conjugated polymers with these nanomaterials can potentially lead to improved sensitivity in optical biosensors.
Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy
Authors: Zeri Ana Carolina , Mesleh Michael F. , Nevzorov Alexander A. , Opella Stanley J.
Published: May 2003

The atomic resolution structure of fd coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles differs from that previously determined by x-ray fiber diffraction. Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38, where there is a distinct kink, and then a straight helix with a different orientation between residues 39 and 49. Residues 1–5 have been shown to be mobile and unstructured, and proline 6 terminates the helix. The structure of the coat protein in virus particles, in combination with the structure of the membrane-bound form of the same protein in bilayers, also recently determined by solid-state NMR spectroscopy, provides insight into the viral assembly process. In addition to their roles in molecular biology and biotechnology, the filamentous bacteriophages continue to serve as model systems for the development of experimental methods for determining the structures of proteins in biological supramolecular assemblies. New NMR results include the complete sequential assignment of the two-dimensional polarization inversion spin-exchange at the magic angle spectrum of a uniformly
15
N-labeled 50-residue protein in a 1.6 × 10
7
Da particle in solution, and the calculation of the three-dimensional structure of the protein from orientational restraints with an accuracy equivalent to an rms deviation of ≈1Å.
Myocardial recovery from ischemia is impaired in CD36-null mice and restored by myocyte CD36 expression or medium-chain fatty acids
Authors: Irie Hiroshi , Krukenkamp Irvin B. , Brinkmann Joep F. F. , Gaudette Glenn R. , Saltman Adam E. , Jou William , Glatz Jan F. C. , Abumrad Nada A. , Ibrahimi Azeddine
Published: May 2003

Long-chain fatty acid uptake, which provides a large part of myocardial energy, is impaired in human and murine hearts deficient in the membrane fatty acid translocase, FAT/CD36. We examined myocardial function in CD36-null mice using the working heart. Fatty acid oxidation and stores of glycogen, triglycerides, and ATP were reduced in CD36-deficient hearts and were restored to WT levels by rescue of myocyte CD36. Under normal perfusion conditions, CD36-null hearts had similar cardiac outputs and end-diastolic pressures as WT or transgenic hearts. After 6 min of ischemia, cardiac output decreased by 41% and end diastolic pressure tripled for CD36-null hearts, with no significant changes in WT or transgenic hearts. Null hearts also failed more frequently after ischemia as compared with WT or transgenics. To dissect out contribution of fatty acid uptake, a perfusate-lacking fatty acids was used. This decreased cardiac output after ischemia by 30% in WT hearts as compared with 50% for CD36-deficient hearts. End diastolic pressure, a negative index of myocardial performance, increased after ischemia in all heart types. Addition to the perfusate of a medium-chain fatty acid (caprylic acid) that does not require CD36 for uptake alleviated poor ischemic tolerance of CD36-null hearts. In summary, recovery from ischemia is compromised in CD36-deficient hearts and can be restored by CD36 rescue or by supplying medium-chain fatty acids. It would be important to determine whether the findings apply to the human situation where polymorphisms of the CD36 gene are relatively common.
Targeting Ras signaling through inhibition of carboxyl methylation: An unexpected property of methotrexate
Authors: Winter-Vann Ann M. , Kamen Barton A. , Bergo Martin O. , Young Stephen G. , Melnyk Stepan , James S. Jill , Casey Patrick J.
Published: May 2003

The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (
1
), methotrexate is known to increase homocysteine (
2
), a compound associated with an elevated risk of heart disease, Alzheimer's disease (
3
), and neural tube defects (
4
). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in
S
-adenosylhomocysteine (
5
), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (
6
), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (
7
), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.
T helper type 1 lymphocytes drive inflammation in human atherosclerotic lesions
Authors: Benagiano Marisa , Azzurri Annalisa , Ciervo Alessandra , Amedei Amedeo , Tamburini Carlo , Ferrari Mauro , Telford John L. , Baldari Cosima T. , Romagnani Sergio , Cassone Antonio , D'Elios Mario M. , Del Prete Gianfranco
Published: May 2003

Atherosclerotic lesions are infiltrated by macrophages and T lymphocytes, potentially reactive to pathogens. We studiedin vivoactivated T lymphocytes that infiltrate atherosclerotic plaques ofHelicobacter pylori-infected patients with or without anti-Chlamydia pneumoniaeantibodies. In all atherosclerotic lesions, T helper type 1 (Th1) cells were predominant.C. pneumoniae-specific T cells were detected only in the plaques of anti-C. pneumoniaeseropositive patients, whereasH. pylori-specific T cells were found in the gastric mucosa but not in the plaques of the same patients. Plaque-derived Th1 cells expressed cytotoxicity, proapoptotic activity, and help for monocyte tissue factor production. Although multifactorial, atherosclerosis can be regarded as a Th1-driven immunopathological condition.
The basic helix–loop–helix–PAS protein ARNT functions as a potent coactivator of estrogen receptor-dependent transcription
Authors: Brunnberg Sara , Pettersson Katarina , Rydin Elin , Matthews Jason , Hanberg Annika , Pongratz Ingemar
Published: May 2003

The biological effects of estrogens are mediated by the estrogen receptors ERα and ERβ. These receptors regulate gene expression through binding to DNA enhancer elements and subsequently recruiting factors such as coactivators that modulate their transcriptional activity. Here we show that ARNT (aryl hydrocarbon receptor nuclear translocator), the obligatory heterodimerization partner for the aryl hydrocarbon receptor and hypoxia inducible factor 1α, functions as a potent coactivator of ERα- and ERβ- dependent transcription. The coactivating effect of ARNT depends on physical interaction with the ERs and involves the C-terminal domain of ARNT and not the structurally conserved basic helix–loop–helix and PAS (Per-ARNT-Sim) motifs. Moreover, we show that ARNT/ER interaction requires the E
2
-activated ligand binding domain of ERα or ERβ. These observations, together with the previous role of ARNT as an obligatory partner protein for conditionally regulated basic helix–loop–helix–PAS proteins like the aryl hydrocarbon receptor or hypoxia inducible factor 1α, expand the cellular functions of ARNT to include regulation of ERα and ERβ transcriptional activity. ARNT was furthermore recruited to a natural ER target gene promoter in a estrogen-dependent manner, supporting a physiological role for ARNT as an ER coactivator.
Different cofactor activities in γ-secretase assembly
Authors: Hu Yue , Fortini Mark E.
Published: May 2003

The γ-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative γ-secretase components have been identified—presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in γ-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in γ-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during γ-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in γ-secretase assembly and activation.
Negative Charges in the Transmembrane Domains of the HERG K Channel Are Involved in the Activation- and Deactivation-gating Processes
Authors: Liu Jie , Zhang Mei , Jiang Min , Tseng Gea-Ny
Published: May 2003

The transmembrane domains of HERG (S1–S3) contain six negative charges: three are conserved in all voltage-gated K channels (D456 and D466 in S2, D501 in S3) and three are unique to the EAG family (D411 in S1, D460 in S2, and D509 in S3). We infer the functional role of these aspartates by studying how substituting them with cysteine, one at a time, affects the channel function. D456C is not functional, suggesting that this negative charge may play a critical role in channel protein folding during biogenesis, as has been shown for its counterpart in the Shaker channel. Data from the other five functional mutants suggest that D411 can stabilize the HERG channel in the closed state, while D460 and D509 have the opposite effect. D466 and D501 both may contribute to voltage-sensing during the activation process. On the other hand, all five aspartates work in a concerted fashion in contributing to the slow deactivation process of the HERG channel. Accessibility tests of the introduced thiol groups to extracellular MTS reagents indicate that water-filled crevices penetrate deep into the HERG protein core, reaching the cytoplasmic halves of S1 and S2. At these deep locations, accessibility of 411C and 466C to the extracellular aqueous phase is voltage dependent, suggesting that conformational changes occur in S1 and S2 or the surrounding crevices during gating. Increasing extracellular [H+] accelerates HERG deactivation. This effect is suppressed by substituting the aspartates with cysteine, suggesting that protonation of these aspartates may contribute to the signaling pathway whereby external [H+] influences conformational changes in the channel's cytoplasmic domains (where deactivation takes place). There is no evidence for a metal ion binding site coordinated by negative charges in the transmembrane domains of HERG, as the one described for the EAG channel.
The Influence of Bag2 and Chain Intrafusal Muscle Fibres on Secondary Spindle Afferents in the Cat
Authors: Durbaba R. , Taylor A. , Ellaway P. H. , Rawlinson S.
Published: May 2003

Static γ‐motor activity is strongly modulated by a particular phase relationship to the cyclic movements of locomotion, and this has a profound effect on the firing patterns of muscle spindle afferents. Whilst primary afferents are affected by both static and dynamic γ‐motor output, secondary afferents are affected significantly only by the static system acting via the intrafusal bag2 and chain fibres. It is therefore important to know how fluctuating patterns of static γ‐motor activity affect secondary afferents and to relate this to the actions of bag2 and chain fibres. We have studied the action of single static γ axons on secondary afferents in cat hindlimb muscles. Various physiological methods were explored to identify which of the intrafusal muscle fibres were being activated in each case, including the use of random stimulation and ramp frequency stimulation. The effects were also recorded of 1 Hz sinusoidally frequency‐modulated γ‐axon stimuli and the amplitude and phase of the resulting afferent modulation related to the involvement of the bag2 and chain fibres. It was found that bag2 fibres are effective in biasing the secondary discharge, but their modulating action is relatively weak and involves a marked phase lag. Chain fibres acting alone cause strong modulation with very little phase lag. Mixed bag2 and chain‐fibre action is most effective in modulating afferent discharge and causes intermediate values of phase lag. The results are discussed in relation to the control of natural movements and it is concluded that an important function of the static γ motor system is to provide a signal to sum algebraically with the length‐related signal. The results do not suggest that it could also usefully control stretch sensitivity.
The Electrophysiological Properties of Spontaneously Beating Pacemaker Cells Isolated from Mouse Sinoatrial Node
Authors: Cho Hyun‐Sung , Takano Makoto , Noma Akinori
Published: May 2003

In order to examine the phenotypic consequences of genetic manipulation in the function of the sinoatrial (SA) node, it is a prerequisite to record the electrical activities of a single pacemaker cell of the SA node of mouse heart. In the present study, we isolated spontaneously beating pacemaker cells from the SA node by enzymatic digestion. The rate of spontaneous action potential firing was 294 ± 59 min−1 at 33–34 °C. The maximal diastolic potential (MDP) was −56.7 ± 7.4 mV and the overshoot was 22.7 ± 6.2 mV. With hyperpolarizing voltage clamp pulses, the hyperpolarization‐activated, cyclic nucleotide‐sensitive cation current (Ih or If) was recorded. Ih started to activate at −70 to ≈−80 mV, more negative than MDP. The half‐maximal activation of Ih was obtained at −107.9 ± 10.4 mV. The inward‐rectifier K+ current (IK1) was also recorded in spontaneously beating myocytes. However, the amplitude of outward IK1 was negligibly small (9.1 ± 3.4 pA at −60 mV). With depolarization, voltage‐gated Na+ current, L‐type Ca2+ current and T‐type Ca2+ current were consistently observed. The sustained inward current (Ist) was also recorded in spontaneously beating pacemaker cells. E4031‐sensitive, rapidly activating delayed rectifier K+ current (IKr) was activated by depolarization, although the amplitude was no more than 38.3 ± 22.2 pA at 0 mV. The chromanol 293B‐sensitive, slowly activating delayed rectifier K+ current (IKs) was not present. The major repolarizing current was the slowly inactivating, 4‐aminopyridine‐insensitive outward current. We concluded that mouse pacemaker cells possess similar membrane currents, including Ist, to those of other species.
β‐Adrenergic or parasympathetic inhibition, heart rate and cardiac output during normoxic and acute hypoxic exercise in humans
Authors: Hopkins Susan R. , Bogaard Harm J. , Niizeki Kyuichi , Yamaya Yoshiki , Ziegler Michael G. , Wagner Peter D.
Published: May 2003

Acute hypoxia increases heart rate (HR) and cardiac output (Qt) at a given oxygen consumption (V̇O2) during submaximal exercise. It is widely believed that the underlying mechanism involves increased sympathetic activation and circulating catecholamines acting on cardiac β receptors. Recent evidence indicating a continued role for parasympathetic modulation of HR during moderate exercise suggests that increased parasympathetic withdrawal plays a part in the increase in HR and Qt during hypoxic exercise. To test this, we separately blocked the β‐sympathetic and parasympathetic arms of the autonomic nervous system (ANS) in six healthy subjects (five male, one female; mean ±s.e.m. age = 31.7 ± 1.6 years, normoxic maximal V̇O2 (V̇O2,max) = 3.1 ± 0.3 l min−1) during exercise in conditions of normoxia and acute hypoxia (inspired oxygen fraction = 0.125) to V̇O2,max. Data were collected on different days under the following conditions: (1)control, (2) after 8.0 mg propranolol I.V. and (3) after 0.8 mg glycopyrrolate I.V. Qt was measured using open‐circuit acetylene uptake. Hypoxia increased venous [adrenaline] and [noradrenaline] but not [dopamine] at a given V̇O2 (P < 0.05, P < 0.01 and P= 0.2, respectively). HR/V̇O2 and Qt/V̇O2 increased during hypoxia in all three conditions (P < 0.05). Unexpectedly, the effects of hypoxia on HR and Qt were not significantly different from control with either β‐sympathetic or parasympathetic inhibition. These data suggest that although acute exposure to hypoxia increases circulating [catecholamines], the effects of hypoxia on HR and Qt do not necessarily require intact cardiac muscarinic and β receptors. It may be that cardiac α receptors play a primary role in elevating HR and Qt during hypoxic exercise, or perhaps offer an alternative mechanism when other ANS pathways are blocked.
Expression of a muscle‐specific, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice
Authors: Nguyen Hal X. , Tidball James G.
Published: May 2003

Nitric oxide (NO) can function as either a pro‐inflammatory or anti‐inflammatory molecule, depending upon its concentration and the microenvironment in which it is produced. We tested whether muscle‐derived NO affects muscle inflammation and membrane lysis that occur in modified muscle use. Transgenic mice with muscle‐specific over‐expression of neuronal NO synthase (nNOS) were generated in which transgene expression was driven by the human skeletal muscle actin promoter. Transgenic mice and non‐transgenic littermates were subjected to hindlimb muscle unloading followed by reloading, which causes muscle inflammation and membrane lysis. NOS expression decreased in transgenic and non‐transgenic mice during muscle unloading. Muscle inflammation was assessed by immunohistochemistry after 24 h of muscle reloading following 10 days of unloading. Soleus muscles of non‐transgenic mice showed significant increases in the concentrations of neutrophils (4.8‐fold) and macrophages (11.3‐fold) during reloading, compared to mice that experienced unloading only. Muscles of transgenic mice showed 51 % fewer neutrophils in reloaded muscles than those of non‐transgenic mice, but macrophage concentrations did not differ from non‐transgenic mice. Muscle membrane damage was determined by measuring influx of an extracellular marker dye. Significantly more membrane damage occurred in muscles of non‐transgenic mice experiencing reloading than in ambulatory controls. However, membrane damage in the reloaded muscles of transgenic mice did not differ from that in ambulatory mice. In vitro cytotoxicity assays confirmed that mouse neutrophils lyse muscle cell membranes, and showed that inhibition of NOS in muscle and neutrophil co‐cultures significantly increased neutrophil‐mediated lysis of muscle cells. Together, these data show that muscle‐derived NO can function as an anti‐inflammatory molecule in muscle that experiences modified loading, and that NO can prevent neutrophil‐mediated damage of muscle cell membranes in vivo and in vitro.
pH‐dependent and ‐independent effects inhibit Ca2+‐induced Ca2+ release during metabolic blockade in rat ventricular myocytes
Authors: O'Neill S. C. , D. A. Eisner
Published: May 2003

We have investigated the role of changes of intracellular pH (pHi) in the effects of metabolic blockade (cyanide plus 2‐deoxyglucose) on Ca2+ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pHi and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca2+ waves, an effect previously shown to be due to inhibition of Ca2+ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pHi and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pHi and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pHi. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pHi but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca2+ release from the SR.
Transmission Dynamics of the Etiological Agent of SARS in Hong Kong: Impact of Public Health Interventions
Authors: Riley Steven , Fraser Christophe , Donnelly Christl A. , Ghani Azra C. , Abu-Raddad Laith J. , Hedley Anthony J. , Leung Gabriel M. , Ho Lai-Ming , Lam Tai-Hing , Thach Thuan Q. , Chau Patsy , Chan King-Pan , Lo Su-Vui , Leung Pak-Yin , Tsang Thomas , Ho William , Lee Koon-Hung , Lau Edith M. C. , Ferguson Neil M. , Anderson Roy M.
Published: May 2003

We present an analysis of the first 10 weeks of the severe acute respiratory syndrome (SARS) epidemic in Hong Kong. The epidemic to date has been characterized by two large clusters—initiated by two separate “super-spread” events (SSEs)—and by ongoing community transmission. By fitting a stochastic model to data on 1512 cases, including these clusters, we show that the etiological agent of SARS is moderately transmissible. Excluding SSEs, we estimate that 2.7 secondary infections were generated per case on average at the start of the epidemic, with a substantial contribution from hospital transmission. Transmission rates fell during the epidemic, primarily as a result of reductions in population contact rates and improved hospital infection control, but also because of more rapid hospital attendance by symptomatic individuals. As a result, the epidemic is now in decline, although continued vigilance is necessary for this to be maintained. Restrictions on longer range population movement are shown to be a potentially useful additional control measure in some contexts. We estimate that most currently infected persons are now hospitalized, which highlights the importance of control of nosocomial transmission.
Role of the Human Cytomegalovirus Major Immediate-Early Promoter's 19-Base-Pair-Repeat Cyclic AMP-Response Element in Acutely Infected Cells
Authors: Keller M. J. , Wheeler D. G. , Cooper E. , Meier J. L.
Published: May 2003

Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.
Lectin-Mediated Retention of p62 Facilitates p62-E1 Heterodimerization in Endoplasmic Reticulum of Semliki Forest Virus-Infected Cells
Authors: Andersson Helena , Garoff Henrik
Published: May 2003

The Semliki Forest virus (SFV) spike subunits p62 and E1 are made from a common coding unit in the order p62-E1. The proteins are separated by the host signal peptidase upon translocation into the endoplasmic reticulum (ER). Shortly thereafter, p62 and E1 form heterodimers. Heterodimerization preferentially occurs between subunits derived from the same translation product, so-called
cis
heterodimerization. As the p62 protein has the capacity to leave the ER in the absence of E1, it has been postulated that there exists a retention mechanism for the p62 protein, putatively at or near the translocon, in the ER in order to promote
cis
heterodimerization (B. U. Barth and H. Garoff, J. Virol.
71:
7857-7865, 1997). Here we show that there exists such a mechanism, that it is at least in part mediated by the ER chaperones calnexin and calreticulin, and that the retention is important for efficient
cis
heterodimerization.
Specific Inhibition of Bovine Viral Diarrhea Virus Replicase
Authors: Sun Jin-Hua , Lemm Julie A. , O'Boyle Donald R. , Racela Jason , Colonno Richard , Gao Min
Published: May 2003

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is ∼2.2 μM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 μM compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 μM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.
Characterization of a Novel Simian Immunodeficiency Virus (SIVmonNG1) Genome Sequence from a Mona Monkey (Cercopithecus mona)
Authors: Barlow Katrina L. , Ajao Adebowale Oluwafemi , Clewley Jonathan P.
Published: May 2003

A novel simian immunodeficiency virus (SIV) sequence has been recovered from RNA extracted from the serum of a mona monkey (Cercopithecus mona) wild born in Nigeria. The sequence was obtained by using novel generic (degenerate) PCR primers and spans from two-thirds into thegaggene to the 3′ poly(A) tail of the SIVmonNG1 RNA genome. Analysis of the open reading frames revealed that the SIVmonNG1 genome codes for a Vpu protein, in addition to Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef proteins. Previously, only lentiviruses infecting humans (human immunodeficiency virus type 1 [HIV-1]) and chimpanzees (SIVcpz) were known to have avpugene; more recently, this has also been found in SIVgsn fromCercopithecus nictitans. Overall, SIVmonNG1 most closely resembles SIVgsn: theenvgene sequence groups with HIV-1/SIVcpzenvsequences, whereas thepolgene sequence clusters closely with thepolsequence of SIVsyk fromCercopithecus albogaris. By bootscanning and similarity plotting, the first half ofpolresembles SIVsyk, whereas the latter part is closer to SIVcol fromColobus guereza. The similarities between the complex mosaic genomes of SIVmonNG1 and SIVgsn are consistent with a shared or common lineage. These data further highlight the intricate nature of the relationships between the SIVs from different primate species and will be helpful for unraveling these associations.
EBNA1 Partitions Epstein-Barr Virus Plasmids in Yeast Cells by Attaching to Human EBNA1-Binding Protein 2 on Mitotic Chromosomes
Authors: Kapoor Priya , Frappier Lori
Published: May 2003

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in
Saccharomyces cerevisiae
and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in
S. cerevisiae
, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in
S. cerevisiae
can occur through chromosome attachment.
A Novel Human Antibody against Human Immunodeficiency Virus Type 1 gp120 Is V1, V2, and V3 Loop Dependent and Helps Delimit the Epitope of the Broadly Neutralizing Antibody Immunoglobulin G1 b12
Authors: Zwick Michael B. , Kelleher Robert , Jensen Richard , Labrijn Aran F. , Wang Meng , Quinnan Gerald V. , Parren Paul W. H. I. , Burton Dennis R.
Published: May 2003

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n= 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.
Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 That Contribute to Heparan Sulfate Proteoglycan Binding
Authors: Opie Shaun R. , Warrington, Kenneth H. , Agbandje-McKenna Mavis , Zolotukhin Sergei , Muzyczka Nicholas
Published: May 2003

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.
Prime-Boost Immunization Schedules Based on Influenza Virus and Vaccinia Virus Vectors Potentiate Cellular Immune Responses against Human Immunodeficiency Virus Env Protein Systemically and in the Genitorectal Draining Lymph Nodes
Authors: Gherardi M. Magdalena , Nájera José Luis , Pérez-Jiménez Eva , Guerra Susana , García-Sastre Adolfo , Esteban Mariano
Published: May 2003

Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8+T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 104PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 107PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8+T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8+T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.
Progressive Truncations C Terminal to the Membrane-Spanning Domain of Simian Immunodeficiency Virus Env Reduce Fusogenicity and Increase Concentration Dependence of Env for Fusion
Authors: Lin Xiaoxu , Derdeyn Cynthia A. , Blumenthal Robert , West John , Hunter Eric
Published: May 2003

The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event.
Cross-Reactivity of HLA-B
*
1801-Restricted T-Lymphocyte Clones with Target Cells Expressing Variants of the Human Cytomegalovirus 72kDa-IE1 Protein
Authors: Prod'homme Virginie , Retière Christelle , Valtcheva Ralitza , Bonneville Marc , Hallet Marie-Martine
Published: May 2003

The impact of natural polymorphism in a cytomegalovirus-dominant HLA-B
*
1801-restricted epitope, IE1
199-206
, on the specific responses of T-cell clones was assessed by measuring their cytolytic activity against target cells expressing mutated recombinant IE1 proteins. Our results suggest an in vivo selection of T lymphocytes that cross-react with multiple IE1 variants.
A diminutive temnospondyl amphibian from the Pennsylvanian of Illinois
Authors: Godfrey Stephen
Published: May 2003

A siderite concretion from the Middle Pennsylvanian, Francis Creek Shale (Carbondale Formation, Desmoinesian Series, Westphalian C), in the Mazon Creek area, WillKankakee counties, Illinois, U.S.A., preserves a hitherto unknown temnospondyl amphibian. The natural mold of the dorsoventrally compressed skeleton (total preserved length of the larva is 38.5 mm (~1.5 inches)), includes a proportionately large skull, hyobranchial denticles, ossified portions of the axial skeleton, elements of both the pectoral and pelvic limbs, and endocasts of segments of the digestive system. Differential staining of the entombing matrix outlines gill filaments, portions of the body wall, and apparently most of the length of the tail. Although this single specimen probably represents a new taxon, too few diagnostic characters are displayed by the larva to name a new species. Large interpterygoid vacuities support its inclusion in the Temnospondyli. Vomers contributing to the formation of the anterior margin of these vacuities place this taxon above the edopoid grade of organization. Furthermore, a very wide cultriform process of the parasphenoid places this taxon above dendrerpetontids. Specific characters that would ally this taxon with any known family have not been identified.
Lariosaurus xingyiensis(Reptilia: Sauropterygia) from the Triassic of China
Authors: Rieppel Olivier , Jinling Li , Jun Liu
Published: May 2003

The anatomy of a specimen of Lariosaurus xingyiensis (IVPP V 11866) from the Middle Triassic of southern China is described in detail. The specimen ranks among the best preserved of its genus. Specimen IVPP V 11866 is the first unequivocal record of the occurrence of Lariosaurus in the Triassic of southern China, indeed the first record of this genus outside the western Tethyan province. A cladistic analysis using parsimony found IVPP V 11866 to represent the sister taxon of Lariosaurus calcagnii from the Middle Triassic of the southern Alps. This result reinforces the signal for western Tethyan paleobiogeographic relationships of the Triassic marine reptiles from southern China.
The postcranial skeleton, phylogenetic position, and probable lifestyle of the Early Triassic reptile Procolophon trigoniceps
Authors: deBraga Michael
Published: May 2003

A morphological study of the postcranial skeleton of Procolophon trigoniceps from the Lower Triassic of South Africa and Antarctica is undertaken. Procolophon shares a sister-group relationship with the procolophonid Tichvinskia from the Lower Triassic of Russia and is a basal member of Procolophonidae. This clade also includes the enigmatic taxon Sclerosaurus, believed most recently to be a pareiasaur relative. Owenettids form a separate lineage from Procolophonidae and are predominantly restricted to the Permian of both South Africa and Madagascar. A phylogenetically based assessment is considered, in which specialized modern taxa (sand lizards) are compared to their nonfossorial sister clade, allowing for "key innovations" to be identified. A similar comparison between owenettids and procolophonids reveals a number of apparent "key innovations" within procolophonids that are suggestive of a burrowing lifestyle for Procolophon.
Robert Lynn Carroll  an appreciation
Authors: Sues Hans-Dieter , Murray Alison M , Anderson Jason S
Published: May 2003

This issue of the Canadian Journal of Earth Sciences is dedicated to Robert L. Carroll in honour of his extensive contributions to vertebrate paleontology and evolution. This career résumé presents an appreciation of his major contributions and lists his numerous scientific publications.
Late Wisconsinan glaciation of Amund and Ellef Ringnes islands, Nunavut: evidence for the configuration, dynamics, and deglacial chronology of the northwest sector of the Innuitian Ice Sheet
Authors: Atkinson Nigel
Published: May 2003

Geomorphic and chronologic evidence from Amund and Ellef Ringnes islands documents the configuration, dynamics, and collapse of the northwest sector of the Innuitian Ice Sheet. These data record the inundation of the Ringnes Islands by northwestward-flowing ice from divides spanning the alpine and lowland sectors of the Innuitian Ice Sheet. Ice-flow indicators and granite dispersal along eastern Amund Ringnes Island suggest Massey Sound was filled by an ice stream discharging coalescent alpine and lowland ice from Norwegian Bay. In contrast, the interior of Amund Ringnes Island was overridden by predominantly non-erosive, granite-free ice from a divide in the lowland sector of the ice sheet. Glacial landforms on Ellef Ringnes Island record coverage by largely non-erosive ice, but it remains uncertain whether these features relate to northward-flowing lowland ice or a cold-based local ice cap. Deglaciation of the Ringnes Islands commenced ~10 000 14C years ago. Deglacial dates between 9.7 and 9.2 ka BP record the sequential entry of marine fauna along Massey and Hassel sounds, concomitant with the southward retreat of trunk ice towards Norwegian Bay. These data suggest marine-based trunk glaciers were vulnerable to calving during pre-Holocene eustatic sea-level rise. However, deglacial dates from inner embayments indicate that residual ice caps persisted on Amund and Ellef Ringnes islands for 800 to 1400 14C years after retreat of trunk ice from the adjacent marine channels. Lateral meltwater channels record the subsequent retreat of these ice caps, which became increasingly confined within upland valleys after 8.6 ka BP.
A new characiform fish (Teleostei: Ostariophysi) from the Eocene of Tanzania
Authors: Murray Alison M
Published: May 2003

Four specimens of a small fossil fish were collected from the Eocene Mahenge site of Tanzania. The specimens, preserved as part and counterpart natural moulds, are identified, predominantly based on the structure of the caudal skeleton, as members of the Characiformes, probably the sister group to the living African Alestidae. The area just behind the skull, in the two specimens that include this area, is distorted, and therefore it is difficult to identify the bones of the Weberian apparatus, although that structure does appear to be present. The fossil record of characiforms includes few articulated skeletons, with only one other African species previously reported from much younger deposits. The new specimens from Mahenge are described here as a new genus and species, Mahengecharax carrolli.