bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Nuclear Localization Sequences Allow Modulation of 5‐Lipoxygenase Nuclear Import
Authors: Luo Ming , Pang C. W. Mark , Gerken Alison E. , Brock Thomas G.
Published: October 2004

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5‐lipoxygenase (5‐LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5‐LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5‐LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5‐LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5‐LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5‐LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5‐LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell‐specific levels of import of 5‐LO. Since nuclear accumulation is known to affect 5‐LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.
Molecular Partitioning during Host Cell Penetration by Toxoplasma gondii
Authors: Charron Audra J. , Sibley L. David
Published: October 2004

During invasion by Toxoplasma gondii, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole membrane (PVM) by the tight apposition of host and parasite cellular membranes. Previous studies suggested that the basis for the selective partitioning of membrane constituents may be a preference for membrane microdomains, and this hypothesis was herein tested. The partitioning of a diverse group of molecular reporters for raft and nonraft membrane subdomains was monitored during parasite invasion by time‐lapse video or confocal microscopy. Unexpectedly, both raft and nonraft lipid probes, as well as both raft and nonraft cytosolic leaflet proteins, flowed unhindered past the host–parasite junction into the PVM. Moreover, neither a raft‐associated type 1 transmembrane protein nor its raft‐dissociated counterpart accessed the PVM, while a multispanning membrane raft protein readily did so. Considered together with previous data, these studies demonstrate that selective partitioning at the host–parasite interface is a highly complex process, in which raft association favors, but is neither necessary nor sufficient for, inclusion into the T. gondii PVM.
LdARF1 in Trafficking and Structural Maintenance of the trans‐Golgi Cisternal Network in the Protozoan Pathogen Leishmania donovani
Authors: Porter‐Kelley Johanna M. , Gerald Noel J. , Engel Juan C. , Ghedin Elodie , Dwyer Dennis M.
Published: October 2004

Adenosine diphosphate ribosylation factors (ARFs) are small guanosine‐5′‐triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183‐amino acid deduced protein of ∼ 20 kDa. We demonstrated by Southern blot analysis that there are at least two copies of ARF1 in the Ld genome. Moreover, Northern blot analysis revealed that Ld ARF1 is expressed on a 1.35 kb transcript in both the insect vector (promastigotes) and mammalian host (amastigotes) forms of this parasite. Fluorescent microscopy studies using Ld promastigotes episomally transfected with an ARF1::GFP (green fluorescent protein) chimeric construct showed that such chimeras appeared to localize to the Golgi region of these organisms. This observation was verified by immunoelectron microscopy using an anti‐GFP antibody. Such studies also revealed that Ld ARF1::GFP chimeras localized to trans‐Golgi vesicles, the flagellar pocket/reservoir and other vesicles located between the trans‐Golgi network and flagellar pocket in these apically polarized cells. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching experiments revealed both the dynamic binding and releasing activity of Ld ARF1 from the Golgi network in these parasites. Further, episomal expression of a constitutively active (“on”) ARF1 (Q71L mutation) resulted in the aberrant swelling and distended‐structure of the trans‐Golgi cisternae in these cells. These results show that Ld ARF1 is transiently associated with the Golgi network and plays a role in the structural maintenance of this organelle in these important human pathogens.
Unexpected Intracellular Localization of the AMD‐Associated Cystatin C Variant
Authors: Paraoan Luminita , Ratnayaka Arjuna , Spiller Dave G. , Hiscott Paul , White Michael R. H. , Grierson Ian
Published: October 2004

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26‐amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age‐related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full‐length mutant (A25T) precursor cystatin C–enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild‐type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild‐type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.
Pharmacologic Rescue of Conformationally‐Defective Proteins: Implications for the Treatment of Human Disease
Authors: Ulloa‐Aguirre Alfredo , Janovick Jo Ann , Brothers Shaun P. , Conn P. Michael
Published: October 2004

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation‐screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non‐native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain‐ or loss‐of‐function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease‐causing misfolded proteins. Rescue of misfolded, “trafficking‐defective”, but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or “pharmacoperones”) are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native‐type‐like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin‐releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.
The α1 and α6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kinetics
Authors: Fisher Janet L.
Published: October 2004

The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The α family is the largest, with six different subtypes. The α1 and α6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the α1 or α6 subunit was expressed in mammalian cells along with β3 and γ2L subunits and the kinetic properties examined with outside‐out patch recordings. The α1β3γ2L receptors responded to GABA with long‐duration openings organized into multi‐opening bursts. In contrast, channel openings of the α6β3γ2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2‐fold slower for α6β3γ2L, compared with the α1β3γ2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the α1 and α6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3–TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N‐terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore‐lining domains are responsible for the kinetic differences conferred by the α1 and α6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition.
Cell swelling‐induced ATP release is tightly dependent on intracellular calcium elevations
Authors: Boudreault Francis , Grygorczyk Ryszard
Published: October 2004

Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom‐designed flow‐through chambers, we investigated the kinetics of cell swelling‐induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o− cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o− cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 ± 67, 70 ± 22 and 17 ± 2.8 pmol min−1 (106 cells)−1, for A549, 16HBE14o− and NIH/3T3, respectively). The concomitant volume changes of substrate‐attached cells were analysed by a 3‐dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch‐sensitive channels, or with the activation of volume‐sensitive, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid‐inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10°C almost completely abolished A549 cell swelling‐induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium‐dependent exocytosis plays a major role in mechanosensitive ATP release.
Pharyngeal airway wall mechanics using tagged magnetic resonance imaging during medial hypoglossal nerve stimulation in rats
Authors: Brennick Michael J. , Pickup Stephen , Dougherty Lawrence , Cater Jacqueline R. , Kuna Samuel T.
Published: October 2004

To better understand pharyngeal airway mechanics as it relates to the pathogenesis and treatment of obstructive sleep apnoea, we have developed a novel application of magnetic resonance imaging (MRI) with non‐invasive tissue tagging to measure pharyngeal wall tissue motion during active dilatation of the airway. Eleven anaesthetized Sprague‐Dawley rats were surgically prepared with platinum electrodes for bilateral stimulation of the medial branch of the hypoglossus nerve that supplies motor output to the protrudor and intrinsic tongue muscles. Images of the pharyngeal airway were acquired before and during stimulation using a gated multislice, spoiled gradient recalled (SPGR) imaging protocol in a 4.7 T magnet. The tag pulses, applied before stimulation, created a grid pattern of magnetically imbedded dark lines that revealed tissue motion in images acquired during stimulation. Stimulation significantly increased cross‐sectional area, and anteroposterior and lateral dimensions in the oropharyngeal and velopharyngeal airways when results were averaged across the rostral, mid‐ and caudal pharynx (P < 0.001). Customized software for tissue motion‐tracking and finite element‐analysis showed that changes in airway size were associated with ventral displacement of  tissues in the ventral pharyngeal wall in the rostral, mid‐ and caudal pharyngeal regions (P < 0.0032) and ventral displacement of the lateral walls in the mid‐ and caudal regions (P < 0.0001). In addition, principal maximum stretch was significantly increased in the lateral walls (P < 0.023) in a ventral–lateral direction in the mid‐ and caudal pharyngeal regions and principal maximum compression (perpendicular to stretch) was significantly increased in the ventral walls in all regions (P < 0.0001). Stimulation did not cause lateral displacement of the lateral pharyngeal walls at any level. The results reveal that the increase in pharyngeal airway size resulting from stimulation of the medial branch of the hypoglossal nerve is predominantly due to ventral displacement of the ventral and lateral pharyngeal walls.
Concentration polarization of hyaluronan on the surface of the synovial lining of infused joints
Authors: Lu Y. , Levick J. R. , Wang W.
Published: October 2004

Hyaluronan (HA) in joints conserves the lubricating synovial fluid by making trans‐synovial fluid escape almost insensitive to pressure elevation (e.g. effusions, joint flexion). This phenomenon, ‘outflow buffering’, was discovered during HA infusion into the rabbit knee joint cavity. It was also found that HA is partially reflected by the joint lining (molecular sieving), and that the reflected fraction R decreases as trans‐synovial filtration rate Q is increased. It was postulated therefore that outflow buffering is mediated by HA reflection. Reflection creates a HA concentration polarization layer, the osmotic pressure of which opposes fluid loss. A steady‐state, cross‐flow ultrafiltration model was previously used to explain the outflow buffering and negative R‐vs.‐Q relation. However, the steady‐state, cross‐perfusion assumptions restricted the model's applicability for an infused, dead‐end cavity or a non‐infused joint during cyclical motion. We therefore developed a new, non‐steady‐state model which describes the time course of dead‐end, partial HA ultrafiltration. The model describes the progressive build‐up of a HA concentration polarization layer at the synovial surface over time. Using experimental parameter values, the model successfully accounts for the observed negative R‐vs.‐Q relation and shows that the HA reflected fraction (R) also depends on HA diffusivity, membrane area expansion and the synovial HA reflection coefficient. The non‐steady‐state model thus explains existing experimental work, and it is a key stage in understanding synovial fluid turnover in intact, moving, human joints or osteoarthritic joints treated by HA injections.
Melatonin advances the circadian timing of EEG sleep and directly facilitates sleep without altering its duration in extended sleep opportunities in humans
Authors: Rajaratnam Shantha M. W. , Middleton Benita , Stone Barbara M. , Arendt Josephine , Dijk Derk‐Jan
Published: October 2004

The rhythm of plasma melatonin originating from the pineal gland and driven by the circadian pacemaker located in the suprachiasmatic nucleus is closely associated with the circadian (approximately 24 h) variation in sleep propensity and sleep spindle activity in humans. We investigated the contribution of melatonin to variation in sleep propensity, structure, duration and EEG activity in a protocol in which sleep was scheduled to begin during the biological day, i.e. when endogenous melatonin concentrations are low. The two 14 day trials were conducted in an environmental scheduling facility. Each trial included two circadian phase assessments, baseline sleep and nine 16 h sleep opportunities (16.00–08.00 h) in near darkness. Eight healthy male volunteers (24.4 ± 4.4 years) without sleep complaints were recruited, and melatonin (1.5 mg) or placebo was administered at the start of the first eight 16 h sleep opportunities. During melatonin treatment, sleep in the first 8 h of the 16 h sleep opportunities was increased by 2 h. Sleep per 16 h was not significantly different and approached asymptotic values of 8.7 h in both conditions. The percentage of rapid eye movement (REM) sleep was not affected by melatonin, but the percentage of stage 2 sleep and sleep spindle activity increased, and the percentage of stage 3 sleep decreased. During the washout night, the melatonin‐induced advance in sleep timing persisted, but was smaller than on the preceding treatment night and was consistent with the advance in the endogenous melatonin rhythm. These data demonstrate robust, direct sleep‐facilitating and circadian effects of melatonin without concomitant changes in sleep duration, and support the use of melatonin in the treatment of sleep disorders in which the circadian melatonin rhythm is delayed relative to desired sleep time.
NHE2‐mediated bicarbonate reabsorption in the distal tubule of NHE3 null mice
Authors: Bailey Matthew A. , Giebisch Gerhard , Abbiati Thecla , Aronson Peter S. , Gawenis Lara R. , Shull Gary E. , Wang Tong
Published: October 2004

NHE3−/− mice display a profound defect in proximal tubule bicarbonate reabsorption but are only mildly acidotic owing to reduced glomerular filtration rate and enhanced H+ secretion in distal nephron segments. In vivo microperfusion of rat distal tubules suggests that a significant fraction of bicarbonate reabsorption in this nephron segment is mediated by NHE2. Two approaches were used to evaluate the role of distal tubule NHE2 in compensating for the proximal defect of H+ secretion in NHE3−/− mice. First, renal clearance experiments were used to assess the impact of HOE694, an inhibitor with significant affinity for NHE2, on excretion of bicarbonate in NHE3−/− and NHE2−/− mice. Second, in vivo micropuncture and microperfusion were employed to measure the concentration of bicarbonate in early distal tubule fluid and to measure distal bicarbonate reabsorption during a constant bicarbonate load. Our data show that HOE694 had no effect on urinary bicarbonate excretion in NHE3+/+ mice, whereas bicarbonate excretion was higher in NHE3−/− mice receiving HOE694. HOE694 induced a significant increase in bicarbonate excretion in mice given an acute bicarbonate load, but there was no effect during metabolic acidosis. Bicarbonate excretion was not affected by HOE694 in bicarbonate‐loaded NHE2−/− mice. In vivo micropuncture revealed that early distal bicarbonate concentration was elevated in both bicarbonate‐loaded and NHE3−/− mice. Further, microperfusion experiments showed that HOE694‐sensitive bicarbonate reabsorption capacity was higher in acidotic and NHE3 null animals. We conclude that NHE2 contributes importantly to acidification in the distal tubule, and that it plays a major role in limiting urinary bicarbonate losses in states in which a high luminal bicarbonate load is presented to the distal tubule, such as in NHE3 null mice.
Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells
Authors: Shi Juan , Mori Emiko , Mori Yasuo , Mori Masayuki , Li Jishuo , Ito Yushi , Inoue Ryuji
Published: October 2004

We investigated, by using the patch clamp technique, Ca2+‐mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor‐operated Ca2+ entry channels. With nystatin‐perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 μm)‐activated TRPC6 currents (ITRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2o+) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2o+ solely inhibited TRPC7 currents (ITRPC7). Vigorous buffering of intracellular Ca2+ (Ca2i+) under conventional whole‐cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2o+, disclosing fast potentiation (EC50: ∼0.4 mm) and inhibition (IC50: ∼4 mm) of ITRPC6 and fast inhibition (IC50: ∼0.4 mm) of ITRPC7. This inhibition of ITRPC6 and ITRPC7 seems to be associated with voltage‐dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of ITRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of ITRPC6 or its acceleration by Ca2o+ probably involves phosphorylation by calmodulin (CaM)‐dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 μm), coexpression of Ca2+ ‐insesentive mutant CaM, and intracellular perfusion of the non‐hydrolysable ATP analogue AMP‐PNP and a CaMKII‐specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh‐activated TRPC7 channel activity was concentration‐dependently suppressed by nanomolar Ca2i+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of ITRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+−CaM‐dependent and ‐independent mechanisms.
Effect of exercise on protein kinase C activity and localization in human skeletal muscle
Authors: Rose Adam J. , Michell Belinda J. , Kemp Bruce E. , Hargreaves Mark
Published: October 2004

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle ergometer exercise for 40 min at 76 ± 1% the peak pulmonary O2 uptake , with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer‐PKC‐substrate motif antibody it was revealed that exercise increased the serine phosphorylation of a ∼50 kDa protein. There were no changes in conventional PKC (cPKC) or PKCθ activities; however, atypical PKC (aPKC) activity was ∼70% higher at 5 and 40 min, and aPKC expression and Thr410/403 phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCα, PKCδ, PKCθ and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKCθ, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.
Mars' South Polar Ar Enhancement: A Tracer for South Polar Seasonal Meridional Mixing
Authors: Sprague A. L. , Boynton W. V. , Kerry K. E. , Janes D. M. , Hunten D. M. , Kim K. J. , Reedy R. C. , Metzger A. E.
Published: October 2004

The gamma ray spectrometer on the Mars Odyssey spacecraft measured an enhancement of atmospheric argon over southern high latitudes during autumn followed by dissipation during winter and spring. Argon does not freeze at temperatures normal for southern winter (∼145 kelvin) and is left in the atmosphere, enriched relative to carbon dioxide (CO
2
), as the southern seasonal cap of CO
2
frost accumulates. Calculations of seasonal transport of argon into and out of southern high latitudes point to meridional (north-south) mixing throughout southern winter and spring.
Polarization Observations with the Cosmic Background Imager
Authors: Readhead A. C. S. , Myers S. T. , Pearson T. J. , Sievers J. L. , Mason B. S. , Contaldi C. R. , Bond J. R. , Bustos R. , Altamirano P. , Achermann C. , Bronfman L. , Carlstrom J. E. , Cartwright J. K. , Casassus S. , Dickinson C. , Holzapfel W. L. , Kovac J. M. , Leitch E. M. , May J. , Padin S. , Pogosyan D. , Pospieszalski M. , Pryke C. , Reeves R. , Shepherd M. C. , Torres S.
Published: October 2004

Polarization observations of the cosmic microwave background with the Cosmic Background Imager from September 2002 to May 2004 provide a significant detection of the
E
-mode polarization and reveal an angular power spectrum of polarized emission showing peaks and valleys that are shifted in phase by half a cycle relative to those of the total intensity spectrum. This key agreement between the phase of the observed polarization spectrum and that predicted on the basis of the total intensity spectrum provides support for the standard model of cosmology, in which dark matter and dark energy are the dominant constituents, the geometry is close to flat, and primordial density fluctuations are predominantly adiabatic with a matter power spectrum commensurate with inflationary cosmological models.
Crystal Structure of F-93 from
Sulfolobus
Spindle-Shaped Virus 1, a Winged-Helix DNA Binding Protein
Authors: Kraft Paul , Oeckinghaus Andrea , Kümmel Daniel , Gauss George H. , Gilmore John , Wiedenheft Blake , Young Mark , Lawrence C. Martin
Published: October 2004

Sulfolobus
spindle-shaped viruses (SSVs), or
Fuselloviridae
, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH ≤4.0; temperature of ≥70°C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-Å crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.
Further Investigation of Simian Immunodeficiency Virus Vif Function in Human Cells
Authors: Gaddis Nathan C. , Sheehy Ann M. , Ahmad K. Muneer , Swanson Chad M. , Bishop Kate N. , Beer Brigitte E. , Marx Preston A. , Gao Feng , Bibollet-Ruche Frederic , Hahn Beatrice H. , Malim Michael H.
Published: October 2004

Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two
vif
-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.
Mink Epithelial Cell Killing by Pathogenic Murine Leukemia Viruses Involves Endoplasmic Reticulum Stress
Authors: Nanua Suparna , Yoshimura Fayth K.
Published: October 2004

We previously demonstrated that mink cells undergo apoptosis after MCF13 murine leukemia virus (MLV) infection. In this study, we observed that virus-infected mink epithelial cells had significantly larger amounts of steady-state levels of MCF13 MLV envelope precursor protein (gPr80

env

) than did
Mus dunni
fibroblasts, which are resistant to virus-induced cytopathicity. Infection of mink cells with the noncytopathic NZB-9 MLV did not result in the accumulation of gPr80

env

. MCF13 MLV infection of mink cells produced low cell surface expression of envelope glycoprotein and less efficient spread of infectious virus. Western blot analysis of mink epithelial cells infected with MCF13 MLV showed an increase in GRP78/BiP, which was not observed for either mink cells infected with NZB-9 MLV or
M. dunni
fibroblasts infected with MCF13 MLV. MCF13 MLV infection of mink cells also resulted in a significant upregulation of CHOP/GADD153. These results indicate that the accumulation of MCF13 MLV gPr80

env

triggers endoplasmic reticulum stress, which may mediate apoptosis in mink epithelial cells.
A Review on Recent Developments in Syntheses of the Post-Secodine Indole Alkaloids. Part I: The Primary Alkaloid Types
Authors: Hájíček Josef
Published: October 2004

This first part of a planned review on developments in the field of total and formal total synthesis of the post-secodine indole alkaloids concentrates on primary alkaloid types. It reviews the synthesis of secodine, aspidospermane, pseudoaspidospermane and ibogane alkaloids; andranginine is also included. It covers the literature from 1992-1993 up to approximately May 2004. A review with 179 references.
Synthesis of a (1→3)-Linked α-L-Fucose Tetrasaccharide
Authors: Ludewig Michael , Thiem Joachim
Published: October 2004

Orthoester-protected benzyl and methyl 1-thiofucopyranosides could be opened to give fucopyranosides unblocked at position 3. Convergent glycosylations of such donors and acceptors led to the corresponding disaccharides, and their block condensation could be elaborated to give the (1→3)-linked α-L-fucose tetrasaccharide. Compounds of this structure are of interest with regard to the role of fucose in metastasis development in lung tissue.
Synthesis of Linear and Branched Regioisomeric Chitooligosaccharides as Potential Mimetics of Natural Oligosaccharide Ligands of Natural Killer Cells NKR-P1 and CD69 Lectin Receptors
Authors: Rohlenová Anna , Ledvina Miroslav , Šaman David , Bezouška Karel
Published: October 2004

Regioisomer of chitobiose13with β(1→3) glycosidic bond and branched analog of chitotriose25having β(1→4) and β(1→3) glycosidic bonds, were prepared and tested as potential mimetics of natural oligosaccharide ligands for activating lectin receptors NKR-P1A and CD69 of natural killer (NK) cells. The structural requirements of NKR-P1 lectin receptor on effective mimetics of its natural ligands has been discussed. A significant binding activity of the branched trisaccharide25to the receptor CD69 was observed.
Synthesis of Some 1,4-Oxazepanes Fused with 1,6-Anhydro-β-D-hexopyranoses. 3,11,13-Trioxa-7-azatricyclo[8.2.1.02,8]tridecane Derivatives
Authors: Trtek Tomáš , Černý Miloslav , Trnka Tomáš , Buděšínský Miloš , Císařová Ivana
Published: October 2004

Some 1,4-oxazepanes based on 1,6-anhydro-β-D-hexopyranoses were prepared from 1,6:3,4-dianhydro-2-O-tosyl-β-D-galactopyranose (1). Cleavage of its oxirane ring with 3-chloropropanol gave 1,6-anhydro-4-O-(3-chloropropyl)-2-O-tosyl-β-D-glucopyranose (2), which was converted in three steps into 4-O-(3-aminopropyl)-1,6:2,3-dianhydro-β-D-mannopyranose (5). The latter compound underwent intramolecular cyclization to afford 3-amino-1,6-anhydro-3-deoxy-3-N,4-O-(propane-1,3-diyl)-β-D-altropyranose (6) that gave the corresponding quaternary ammonium salt 10 by N-methylation. Acid cleavage of the 1,6-anhydro bond in 7 gave the D-altrose derivative 3-acetamido-3-deoxy-3-N,4-O-(propane-1,3-diyl)-β-D-altropyranose (11).
Kinetics of Methylation of Methyl 5-Deoxy-α/β-D-xylofuranosides
Authors: Oščendová Mária , Moravcová Jitka
Published: October 2004

The kinetics of methylation of methyl 5-deoxy-α-D-xylofuranoside (1), methyl 5-deoxy-β-D-xylofuranoside (2) and their partly methylated derivatives with methyl iodide in the presence of sodium hydroxide in acetonitrile was studied. The reaction rate was independent of the base concentration during the first half-time only and the methylation proceeded as a first-order reaction. The rate constants of all side and consecutive reactions were calculated and the influence of both polar and steric effect is discussed. The methylation of 1 was highly regioselective giving almost exclusively 5-deoxy-2-O-methyl-α-D-xylofuranoside.
Synthesis of N9- and N7-[2-Hydroxy-3-(phosphonomethoxy)propyl] Derivatives of N6-Substituted Adenines, 2,6-Diaminopurines and Related Compounds
Authors: Krečmerová Marcela , Masojídková Milena , Holý Antonín
Published: October 2004

Base-catalyzed reactions of diethyl [(oxiranylmethoxy)methyl]phosphonate (2) with purine bases (adenine, 2,6-diaminopurine, 6-chloropurine and 2-amino-6-chloropurine) gave corresponding 9- or 7-[2-hydroxy-3-(phosphonomethoxy)propyl] purines. The adenine and 2,6-diaminopurine derivatives cyclize to cyclic phosphonates 4 and 6. The 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives of N6-substituted adenine and 2,6-diaminopurine (15-27) were prepared by the treatment of diethyl {[3-(6-chloropurin-9-yl)-2-hydroxypropoxy]methyl}phosphonate (11) or diethyl {[3-(2-amino-6-chloropurin-9-yl)-2-hydroxypropoxy]methyl}phosphonate (13) with primary or secondary amines. The reaction of 6-chloro- or 2-amino-6-chloropurine derivatives (11, 13) with thiourea gave the corresponding diethyl purine-6-thiol or 2-aminopurine-6-thiol phosphonates 47, 48. The guanine derivative 49 was prepared by the treatment of compound 13 with 80% acetic acid. All diethyl phosphonates were transformed to free phosphonic acids (31-43, 50-52) by the action of bromotrimethylsilane and subsequent hydrolysis.
Synthesis of 1,6-Anhydro-2,3,4-trideoxy-2,3-epimino- and 1,6-Anhydro-2,3,4-trideoxy-3,4-epimino-β-D-hexopyranoses and Their NMR and Infrared Spectra
Authors: Karban Jindřich , Buděšínský Miloš , Kroutil Jiří
Published: October 2004

A complete series of 2,3,4-trideoxy-2,3-epimino and 2,3,4-trideoxy-3,4-epimino derivatives of 1,6-anhydro-β-D-hexopyranoses were prepared by lithium aluminium hydride reduction of vicinal trans azido tosylates. Unusual formation of the aziridine ring from precursors with the trans-diequatorial arrangement of the reacting groups was observed. NMR and infrared spectra of the aziridines are discussed.
Covalent Analogues of DNA Base-Pairs and Triplets VII. Synthesis and Cytostatic Activity of Bis(purin-6-yl)acetylene and -diacetylene Nucleosides
Authors: Nauš Petr , Votruba Ivan , Hocek Michal
Published: October 2004

The title bis(purin-6-yl)acetylene and -diacetylene nucleoside derivatives were prepared as covalent base-pair analogues starting from acyl-protected 6-ethynylpurine and 6-iodopurine nucleosides by the Sonogashira cross-coupling or oxidative alkyne-dimerization reactions followed by deprotection. The key starting acyl-protected 6-ethynylpurine nucleosides were prepared by a sequence of cross-coupling reactions of protected 6-halopurine nucleosides with (trimethylsilyl)acetylene followed by a modified desilylation with TBAF in presence of acetic acid. Surprisingly, the acyl-protected nucleosides exhibited significant cytostatic activity higher than the fully deprotected title compounds.
Electronic spectroscopy of the nonlinear carbon chains C4H4+ and C8H4+
Authors: Araki Mitsunori , Cias Pawel , Denisov Alexey , Fulara Jan , Maier John P
Published: October 2004

The electronic spectrum of a nonlinear carbon chain radical C4H4+ was observed after mass-selective deposition in a 6 K neon matrix. The corresponding gas-phase spectra of C4H4+ and C4D4+ have been observed in the 512 to 513 nm region and at 710 nm for C8H4+. These were detected in direct absorption by cavity ringdown spectroscopy through a supersonic planar discharge. The electronic transition energies of these nonlinear carbon chain radicals correlate well with those of the polyacetylene cations HCnH+ (n = 4, 6, 8). The observed profiles are reproduced with rotational constants obtained by ab initio geometry optimizations and extrapolation between the ground and excited electronic states. Key words: nonlinear carbon chain, carbon cation, electronic transition, diffuse interstellar bands, molecular structure.
The BrO bond in halogen oxides  Empirical force constants and electronic characteristics
Authors: Escribano Rafael , Ortega Ismael K , Mosteo Rafael G , Gómez Pedro C
Published: October 2004

A number of bromine oxides and mixed chlorinebromine oxides for which spectroscopic information is available have been chosen to investigate the nature and characteristics of the BrO bond. The study consists of the empirical determination of stretching force constants for these bonds from observed vibrational spectroscopic data and the analysis of the topological characteristics of the bonds via ab initio calculations. The latter have been performed at the MP2 level with a 6-311+G(2df) basis set, to provide a uniform and systematic framework for comparing these species. Three types of BrO bonds have been found, with different characteristics of strength and electron density. The results are compared with those recently found for the ClO bond in chlorine oxides.Key words: bromine oxides, bond electronic structure.
Revisiting lipid  general anesthetic interactions (I): Thinned domain formation in supported planar bilayers induced by halothane and ethanol
Authors: Leonenko Zoya V , Cramb David T
Published: October 2004

A long-standing question in anesthesia is that of the molecular mechanism. Do anesthetics target proteins or change membrane properties or both? We used temperature-dependent magnetic A/C mode atomic force microscopy (AFM) to study interaction of the volatile anesthetics halothane and ethanol with model membranes made from supported planar bilayers (SPBs) of 1,2-dioleoyl-sn-3-glycero-3-phosphocholine (DOPC), dioleoyltrimethylammonium propane (DOTAP), or 1,2-dipalmitoyl-sn-3-glycero-3-phosphocholine (DPPC). We found that the incorporation of halothane or ethanol induces structural changes in the bilayer. These compounds cause thickness reduction in Lα bilayers (either globally or in domains) and the formation of domains with reduced thickness in Lβ phase bilayers. We propose that an anesthetic-induced increased area per lipid drives local chain disorder, thus promoting local phase change. The characteristics of SPBs with halothane or ethanol incorporated were compared with characteristics of the Lα and Lβ phases of anesthetic-free SPBs.Key words: atomic force microscopy, anesthesia, lipid bilayer domains, phase transition
Revisiting lipid  general anesthetic interactions (II): Halothane location and changes in lipid bilayer microenvironment monitored by fluorescence
Authors: Carnini Anna , Phillips Heather A , Shamrakov Leanne G , Cramb David T
Published: October 2004

A universal mechanism for the action of general anesthetics (GA) is not yet available. In this study, we investigated the interaction between halothane and 1,2-dipalmitoyl-sn-3-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-3-glycero-3-phosphocholine (DOPC) bilayers labeled with Laurdan, Prodan, and NBD-C6-PC as the reporter probes using steady-state fluorescence spectroscopy. We have evidence that halothane is located on the acyl chain side, near the headgroup region of the bilayer. Additionally, we find that halothane may be inhomogeneously distributed within DOPC and DPPC bilayers. We also show data that indicate halothane increases the free volume available to fluorescent probes. Differential scanning calorimetry and UV scanning calorimetry experiments were implemented to further observe the effects of halothane addition to the DPPC lipid bilayer. A significant shift of the phase-transition temperature of the DPPC system was observed. Our findings suggest that general anesthetic  lipid bilayer interactions may play a significant role in the overall mechanism of anesthetic action, and these effects should not be ignored when interactions between membrane proteins and anesthetics are considered.Key words: liposomes, anesthesia, fluorescence, phase transition, phospholipid bilayers.
The Canadian Light Source  History and scientific prospects
Authors: Bancroft G M
Published: October 2004

The Canadian Light Source (CLS) in Saskatoon has been under construction for the last 4 years, and will be producing a number of very intense beams of far-IR, IR, soft and hard X-rays in 2004 for use by several hundred Canadian scientists in chemistry, surface and material science, and a host of other scientific disciplines. The CLS will dramatically enhance the Canadian spectroscopic tradition that Gerhard Herzberg help create. I begin this article (from my 2002 CIC Montreal Medal lecture) with an overview of the history of SR in Canada, beginning in 1972 with the first Canadian synchrotron workshop organized at the University of Western Ontario by Bill McGowan, and attended by Dr. Herzberg. The CLS facility is then described, along with the properties of the first and second set of beamlines to be built at the CLS. These SR beams, in the IR and X-ray regions from the third generation CSL ring, will be competitive in brightness and intensity with the best beamlines in the world for most applications. Finally, some of the present Canadian SR research at foreign SR sources is described across the entire SR spectrum. All known spectroscopic and diffraction experiments are dramatically enhanced with SR; and SR opens up new areas of spectroscopy, microscopy, and spectromicroscopy that cannot be studied with any other source of radiation.Key words: synchrotron light, X-rays, infrared, spectroscopy.
The spectrum of singlet SiH2
Authors: Yurchenko S N , Bunker P R , Kraemer W P , Jensen P
Published: October 2004

We report a theoretical study of the two lowest singlet electronic states ([Formula: see text]1A1 and Ã1B1) of silylene SiH2. These states become degenerate as a 1Δg state at linear configurations and are subject to the Renner effect. In ab initio calculations we have determined the potential energy and dipole moment surfaces for each state, and the transition moment surface between the states. Parameterized analytical functions have been fitted through the various sets of ab initio points, and the parameter values obtained for the potential energy surfaces have been further refined in fittings to experimental spectroscopic data. In these latter fittings, we use as input data experimentally derived energy differences together with ab initio points. In this manner, we achieve refined potential energy surfaces that behave reasonably also in regions of configuration space that are not sampled by the wavefunctions of the states for which experimentally derived energies are available. The calculation of rovibronic energies, the fittings to experimentally derived energies, and simulations of Ã1B1 → [Formula: see text]1A1 emission spectra of SiH2 have been carried out with the RENNER program system. The higher excited vibrational states of [Formula: see text]1A1 SiH2 form polyads of heavily interacting states and many polyad states have been observed in dispersed fluorescence studies. The present theoretical work shows that owing to the heavy interaction between the states in the polyads, it is difficult to obtain unambiguous assignments for them.Key words: silylene, RENNER, ab initio.PACS Nos.: 31.15.Ar, 33.20.Wr, 33.20.Ea
High-resolution terahertz spectrum of CH2  Low J rotational transitions near 2 THz
Authors: Brünken S , Michael E A , Lewen F , Giesen Th. , Ozeki H , Winnewisser G , Jensen P , Herbst E
Published: October 2004

The methylene radical (CH2) was very important to Gerhard Herzberg. We have carried out high-resolution spectroscopic measurements on two energetically low-lying, pure rotational transitions of methylene in its ground vibrationalelectronic state at frequencies near 2 THz. One of the transitions  the NKaKc = 211 ← 202 multiplet  belongs to ortho-CH2 and is centered at 1.954 THz. The other rotational transition  the NKaKc = 110 ← 101 multiplet  belongs to para-CH2 and is centered at 1.915 THz. Since the ground electronic state of methylene has 3B1 symmetry, the rotational transitions are split into three fine-structure components, while the ortho transitions are split additionally by hyperfine structure. In total, we have measured 29 new lines, six for the para transition and the remainder for the ortho transition. The newly measured lines can be added to high-resolution spectral data obtained by ourselves and others at lower frequencies. Progress towards a global fit is discussed. Key words: spectroscopy, THz frequencies, radicals, methylene, interstellar medium.