bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining the function of xeroderma pigmentosum group F protein in psoralen interstrand cross-link-mediated DNA repair and mutagenesis
Authors: CHEN Zhiwen , XU Xiaoxin Susan , HARRISON Jason , WANG Gan
Published: April 2004

Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167→A transversions, whereas most of the mutations generated in the XPF cells were T167→G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167→A mutations were increased and the T167→G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis.
Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP
Authors: SIMMONS Christine Q. , SIMMONS Alan J. , HAUBNER Aaron , REAM Amber , DAVIDSON Jeffrey N.
Published: April 2004

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-α-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992→Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954→Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch–Nyhan syndrome.
Slow calcium oscillations in human spermatozoa
Authors: KIRKMAN-BROWN Jackson C. , BARRATT Christopher L. R. , PUBLICOVER Stephen J.
Published: April 2004

We have used single-cell imaging to investigate intracellular Ca2+ signalling in human spermatozoa stimulated with progesterone (3 µM). In approx. 9% of cells progesterone caused the activation of slow repetitive [Ca2+]i (intracellular Ca2+ concentration) oscillations, with a period of 1–4 min, which persisted for the duration of recording (20–30 min). Pretreatment with nifedipine, which blocks T- and L-type voltage-operated Ca2+ channels in spermatogenic cells, did not modify the characteristics of the oscillations, but reduced the proportion of cells in which they were observed. Stimulation with Bay K 8644 or FPL64176 induced [Ca2+]i oscillations in 5–10% of cells, but their frequency was low (period, 4–5 min). Application of valinomycin (1 µM) to clamp membrane potential at EK (equilibrium potential for potassium) did not modify activity in oscillating cells, showing that plasma membrane potential and activation of voltage-operated conductances are not involved in the mechanism by which sperm [Ca2+]i oscillations are generated.
Expression of pro-inflammatory markers by human dermal fibroblasts in a three-dimensional culture model is mediated by an autocrine interleukin-1 loop
Authors: KESSLER-BECKER Daniela , KRIEG Thomas , ECKES Beate
Published: April 2004

In vivo, fibroblasts reside in connective tissues, with which they communicate in a reciprocal way. Such cell–extracellular matrix interactions can be studied in vitro by seeding fibroblasts in collagen lattices. Depending upon the mechanical properties of the system, fibroblasts are activated to assume defined phenotypes. In the present study, we examined a transcriptional profile of primary human dermal fibroblasts cultured in a relaxed collagen environment and found relative induction (>2-fold) of 393 out of approx. 7100 transcripts when compared with the same system under mechanical tension. Despite down-regulated proliferation and matrix synthesis, cells did not become generally quiescent, since they induced transcription of numerous other genes including matrix metalloproteinases (MMPs) and growth factors/cytokines. Of particular interest was the induction of gene transcripts encoding pro-inflammatory mediators, e.g. cyclo-oxygenase-2 (COX-2), and interleukins (ILs)-1 and -6. These are apparently regulated in a hierarchical fashion, since the addition of IL-1 receptor antagonist prevented induction of COX-2, IL-1 and IL-6, but not that of MMP-1 or keratinocyte growth factor (KGF). Our results suggest strongly that skin fibroblasts are versatile cells, which adapt to their extracellular environment by displaying specific phenotypes. One such phenotype, induced by a mechanically relaxed collagen environment, is the ‘pro-inflammatory’ fibroblast. We propose that fibroblasts that are embedded in a matrix environment can actively participate in the regulation of inflammatory processes.
Comparative analysis of DNA methylation patterns in transgenic Drosophila overexpressing mouse DNA methyltransferases
Authors: MUND Cora , MUSCH Tanja , STRÖDICKE Martin , ASSMANN Birte , LI En , LYKO Frank
Published: April 2004

DNA methyltransferases (Dnmts) mediate the epigenetic modification of eukaryotic genomes. Mammalian DNA methylation patterns are established and maintained by co-operative interactions among the Dnmt proteins Dnmt1, Dnmt3a and Dnmt3b. Owing to their simultaneous presence in mammalian cells, the activities of individual Dnmt have not yet been determined. This includes a fourth putative Dnmt, namely Dnmt2, which has failed to reveal any activity in previous assays. We have now established transgenic Drosophila strains that allow for individual overexpression of all known mouse Dnmts. Quantitative analysis of genomic cytosine methylation levels demonstrated a robust Dnmt activity for the de novo methyltransferases Dnmt3a and Dnmt3b. In addition, we also detected a weak but significant activity for Dnmt2. Subsequent methylation tract analysis by genomic bisulphite sequencing revealed that Dnmt3 enzymes preferentially methylated CpG dinucleotides in a processive manner, whereas Dnmt2 methylated isolated cytosine residues in a non-CpG dinucleotide context. Our results allow a direct comparison of the activities of mammalian Dnmts and suggest a significant functional specialization of these enzymes.
Inosine 5′-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo
Authors: McLEAN Jeremy E. , HAMAGUCHI Nobuko , BELENKY Peter , MORTIMER Sarah E. , STANTON Martin , HEDSTROM Lizbeth
Published: April 2004

Inosine 5´-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione β-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.
Augmented intrinsic activity of Factor VIIa by replacement of residues 305, 314, 337 and 374: evidence of two unique mutational mechanisms of activity enhancement
Authors: PERSSON Egon , BAK Helle , ØSTERGAARD Anette , OLSEN Ole H.
Published: April 2004

Coagulation Factor VIIa (FVIIa) lacks the ability to spontaneously complete the conversion to a fully active enzyme after specific cleavage of an internal peptide bond (Arg152–Ile153) in the zymogen. Recently, several variants of FVIIa with enhanced intrinsic activity have been constructed. The in vitro characterization of these variants has shed light on molecular determinants that put restrictions on FVIIa in favour of a zymogen-like conformation and warrants continued efforts. Here we describe a new FVIIa variant with high intrinsic activity containing the mutations Leu305→Val, Ser314→Glu, Lys337→Ala, and Phe374→Tyr. The variant, called FVIIaVEAY, processes a tripeptidyl substrate very efficiently because of an unprecedented, 5.5-fold lowering of the Km value. Together with a 4-fold higher substrate turnover rate this gives the variant a catalytic efficiency 22 times that of wild-type FVIIa, which is reflected in a considerably enhanced susceptibility to inhibition by antithrombin and other inhibitors. For instance, the affinity of FVIIaVEAY for the S1 probe and inhibitor p-aminobenzamidine is represented by an 8-fold lower Ki value compared with that of FVIIa. Activation of Factor X in solution occurs about 10 times faster with FVIIaVEAY than with FVIIa, due virtually exclusively to an increased kcat value. The high activity of FVIIaVEAY is not accompanied by an increased burial of the N-terminus of the protease domain. A comparison of the kinetic parameters and molecular properties of FVIIaVEAY with those of the previously described mutant V158D/E296V/M298Q-FVIIa (FVIIaIIa), and the locations of the substitutions in the two variants, reveals what appear to be two profoundly different structural mechanisms dictating improvements in enzymic performance.
Thrombospondin-1 mediates distal tubule hypertrophy induced by glycated albumin
Authors: YANG Yu-Lin , CHUANG Lea-Yea , GUH Jinn-Yuh , LIU Shu-Fen , HUNG Min-Yuan , LIAO Tung-Nan , HUANG Yu-Lun
Published: April 2004

Diabetic nephropathy is characterized by early hypertrophy in both glomerular and tubuloepithelial elements. However, no studies to date have established a direct causal link between hyperglycaemia and renal hypertrophy. Our previous studies have found that high glucose does not induce cellular hypertrophy or expression of TGF-β1 (transforming growth factor-β1) in distal renal tubule cells [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182–193]. In the present study, we used AGEs (advanced glycation end-products) to mimic long-term hyperglycaemia. Similar to glucose, AGEs did not induce TGF-β1 mRNA in distal renal tubule cells [MDCK (Madin–Darby canine kidney) cells]; however, TGF-β1 bioactivity was increased significantly. This result indicated post-translational regulation. Since TSP-1 (thrombospondin-1) has been demonstrated to activate latent TGF-β1 in a variety of systems, the following experiments were performed. We found that AGEs dose-dependently increased both intracellular and extracellular levels of TSP-1. Purified TSP-1, like AGEs, increased the cellular protein content. Furthermore, anti-TSP-1 neutralizing antibodies attenuated the AGE-induced increase in TGF-β1 bioactivity and hypertrophy. Thus TSP-1 might mediate AGE-induced distal renal tubule hypertrophy. In addition, we observed several putative transcription factor binding sites in the TSP-1 promoter, including those for AP-1 (activator protein-1), CREB (cAMP response element binding protein), NF-κB (nuclear factor-κB), SRF (serum response factor) and HSF (heat-shock factor), by sequence mapping. We used an enhancer assay to screen possible transcription factors involved. We showed that AP-1 and CREB were specifically induced by AGEs; furthermore, TFD (transcription factor decoy) for AP-1 could attenuate the AGE-induced increases in TSP-1 levels and cellular hypertrophy. Thus regulation of TSP-1 might be critical for hyperglycaemic distal tubule hypertrophy. Furthermore, TSP-1 TFD might be a potential approach to ameliorate diabetic renal hypertrophy.
Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha
Authors: KEUM Eunyoung , KIM Yeonhee , KIM Jungyean , KWON Soojin , LIM Yangmi , HAN Innoc , OH Eok-Soo
Published: April 2004

During cell–matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-α (protein kinase C-α) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133–8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-α in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-α. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-α and enhanced the translocation of PKC-α into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-α in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-α immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-α. Taken together, these results suggest that syndecan-4 interacts with PKC-α in vivo and regulates its localization, activity and stability.
Hormone-sensitive lipase - new roles for an old enzyme
Authors: YEAMAN Stephen J.
Published: April 2004

Although described initially as an intracellular adipocyte-specific triacylglycerol lipase, it is now clear that HSL (hormone-sensitive lipase) is expressed in multiple tissues and plays a number of roles in lipid metabolism, including that of a neutral cholesteryl ester hydrolase. The major isoform is a single polypeptide with a moleclar mass of approx. 84 kDa and which comprises three major domains: a catalytic domain, a regulatory domain encoding several phosphorylation sites and an N-terminal domain involved in protein–protein and protein–lipid interactions. The activity of HSL is regulated acutely by several mechanisms, including reversible phosphorylation by a number of different protein kinases, translocation to different sites within the cell and interaction with a number of proteins, some of which may serve to direct the inhibitory products of HSL away from the protein. It is also apparent from work with HSL null mice that more than one enzyme species may be classified as a hormone-sensitive lipase. The possible presence of HSL in macrophages remains controversial, and the role of the protein in pancreatic β-cells has yet to be fully elucidated. Altered expression of HSL in different cell types may be associated with a number of pathological states, including obesity, atherosclerosis and Type II diabetes.
Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells
Authors: DE BOSSCHER Karolien , HILL Caroline S. , NICOLÁS Francisco J.
Published: April 2004

Smad4 is an essential signal transducer of the transforming growth factor β (TGF-β) signalling pathway and has been identified as a tumour suppressor, being mutated in approx. 50% of pancreatic cancers and approx. 15% of colorectal cancers. Two missense mutations in the C-terminal domain of Smad4, D351H (Asp351→His) and D537Y (Asp537→Tyr), have been described recently in the human colorectal cancer cell lines CACO-2 and SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell, Wasan, Roylance, Bodmer and Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9719–9723]. Previous work in vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu, Fairman, Penry and Shi (2001) J. Biol. Chem. 276, 20688–20694]. In the present study, we investigate the functional consequences of these point mutations in vivo. We demonstrate that neither of these colorectal cancer cells undergo growth arrest in response to TGF-β, which can be explained, at least in part, by their inability to up-regulate cyclin-dependent kinase inhibitors p21CIP1 or p15INK4b after TGF-β stimulation. Although the point-mutated Smad4s are expressed at normal levels in these colorectal cancer cells, they cannot interact with either TGF-β-induced phosphorylated Smad2 or Smad3. As a result, these Smad4 mutants do not accumulate in the nucleus after TGF-β stimulation, are not recruited to DNA by relevant Smad-binding transcription factors and cannot generate transcriptionally active DNA-bound complexes. Therefore both these colorectal tumour cells completely lack functional Smad4 activity owing to the missense mutations. Given the location of these mutations in the three-dimensional structure of the Smad4 C-terminal domain, the results also give us significant insights into Smad complex formation.
Master of all things phosphorylated
Authors: YAFFE Michael B.
Published: April 2004

Serine/threonine phosphorylation plays a central role in cellular regulation, either by altering a protein's activity directly or by inducing specific protein–protein interactions, which, in turn, affect localization, binding specificity or activity. One group of molecules that bind to phosphoserine/phosphothreonine-containing sequences are the 14-3-3 proteins, which regulate a wide range of cellular targets. A new analysis of the 14-3-3 phosphoproteome using affinity chromatography has revealed many previously unknown 14-3-3 ligands whose binding to 14-3-3 proteins is phosphorylation-dependent. This study by the Mackintosh group in this issue of the Biochemical Journal paves the way for future work on these important 14-3-3-interacting proteins.
Haemodynamic effects of hyperbaric hyperoxia in healthy volunteers: an echocardiographic and Doppler study
Authors: MOLÉNAT Florence , BOUSSUGES Alain , GRANDFOND Aliocha , ROSTAIN Jean-Claude , SAINTY Jean-Marie , ROBINET Claude , GALLAND François , MELIET Jean Louis
Published: April 2004

In the present study, we observed the haemodynamic changes, using echocardiography and Doppler, in ten healthy volunteers during 6 h of compression in a hyperbaric chamber with a protocol designed to reproduce the conditions as near as possible to a real dive. Ambient pressure varied from 1.6 to 3 atm (1 atm=101.325 kPa) and partial pressure of inspired O2 from 1.2 to 2.8 atm. Subjects performed periods of exercise with breathing through a closed-circuit self-contained underwater breathing apparatus (SCUBA). Subjects did not eat or drink during the study. Examinations were performed after 15 min and 5 h. After 15 min, stroke volume (SV), left atrial (LA) diameter and left ventricular (LV) end-diastolic diameter (LVEDD) decreased. Heart rate (HR) and cardiac output (CO) did not vary, but indices of the LV systolic performance decreased by 10% and the LV meridional wall stress increased by 17%. After 5 h, although weight decreased, the serum protein concentration increased. Compared with values obtained after 15 min, SV and CO decreased, but LV systolic performance, LA diameter, LVEDD and LV meridional wall stress remained unchanged. Compared with the reference values obtained at sea level, total arterial compliance decreased, HR remained unchanged and CO decreased. In conclusion, hyperbaric hyperoxia results in significant haemodynamic changes. Initially, hyperoxia and the SCUBA system are responsible for reducing LV preload, increasing LV afterload and decreasing LV systolic performance, although CO did not change. Prolonged exposure resulted in a further decrease in LV preload, because of dehydration, and in a further increase in LV afterload, due to systemic vasoconstriction, with the consequence of decreasing CO.
Folic acid does not improve endothelial function in healthy hyperhomocysteinaemic subjects
Authors: WOODMAN Richard J. , CELERMAJER David E. , THOMPSON Peter L. , HUNG Joseph
Published: April 2004

Folic acid supplementation lowers total plasma homocysteine (tHcy) and improves endothelial function in individuals with coronary artery disease (CAD) and in those with additional CAD risk factors. In the present study, we assessed whether endothelial function is impaired in healthy subjects with hyperhomocysteinaemia but without other CAD risk factors and whether folic acid supplementation improves endothelial function in these subjects. Flow-mediated dilatation (FMD) of the brachial artery was performed on 26 healthy subjects, age 49±2 years (mean±S.E.M.), with high tHcy (15.6±1.5 µmol/l) and 16 healthy age-matched subjects with low tHcy (7.9±0.6 µmol/l; P<0.001). Subjects with high tHcy were then randomized to receive 5 mg/day of folic acid or placebo for 8 weeks in a double-blind cross-over trial with a 4-week washout. FMD was not associated with tHcy and was not different between high and low tHcy groups (7.0±0.6% compared with 6.6±1.2%, P=0.76). Treatment with folic acid decreased tHcy by 34% in hyperhomocysteinaemic subjects (P=0.02 compared with placebo), but had no effect on FMD (+0.5±0.6% compared with -0.7±0.5%; P=0.17 compared with placebo). In healthy subjects with hyperhomocysteinaemia, but without additional cardiovascular risk, endothelial function is unimpaired and folic acid supplementation has no additional effect.
Modulation of the liver export protein synthetic response to feeding by an n−3 fatty-acid-enriched nutritional supplement is associated with anabolism in cachectic cancer patients
Authors: BARBER Matthew D. , PRESTON Tom , McMILLAN Donald C. , SLATER Christine , ROSS James A. , FEARON Kenneth C. H.
Published: April 2004

The acute-phase protein response is associated with accelerated weight loss and shortened survival in cancer. This may be due to hepatic protein synthesis increasing demand for amino acids. An n-3 fatty-acid-enriched nutritional supplement will moderate aspects of cachexia in cancer patients. The present study examined the effect of such a supplement on hepatic synthesis of albumin and fibrinogen. Albumin and fibrinogen synthesis were measured in the fed and fasting state in eight weight-losing patients with pancreatic cancer by an intravenous flooding dose technique. Tracer incorporation into proteins was measured by GC/MS. Patients were restudied after 3 weeks of oral supplement enriched with fish oil (providing 2510 kJ/day and 2 g of eicosapentaenoic acid/day). At baseline, all patients were losing weight (median, 2.4 kg/month). After 3 weeks of consumption of the fish-oil-enriched nutritional supplement, patients′ weight stabilized (median change, +1 kg; P=0.01). At baseline, albumin and fibrinogen synthesis rates were stimulated in the fed compared with the fasting state [14.2 compared with 11.3 g/day (29% rise; P=0.01) and 4.5 compared with 3.3 g/day (38% rise; P=0.01) respectively]. After 3 weeks of the supplement, this stimulation in the fed state was no longer observed for albumin and was reduced for fibrinogen [11.2 compared with 10.5 g/day (3% rise; P=0.21) and 3.7 compared with 2.9 g/day (17% rise; P=0.01) respectively]. After 3 weeks, the combined albumin plus fibrinogen synthetic rate tended to fall in the fasting state (14.7 compared with 12.3 g/day; P=0.09) and was significantly reduced in the fed state (18.7 compared with 14.6 g/day; P=0.01). Modulation of hepatic export protein synthesis with feeding may have contributed to the net whole-body anabolism observed with administration of the n-3 fatty-acid-enriched oral supplement.
Increased plasma non-esterified fatty acids and platelet-activating factor acetylhydrolase are associated with susceptibility to atherosclerosis in mice
Authors: SINGH Uma , ZHONG Shumei , XIONG Momiao , LI Tong-bin , SNIDERMAN Allan , TENG Ba-Bie
Published: April 2004

Animal models provide vital tools to explicate the pathogenesis of atherosclerosis. Accordingly, we established two atherosclerosis-prone mice models: (i) mice lacking the LDL (low-density lipoprotein) receptor (LDLR) and the ability to edit apo (apolipoprotein) B mRNA (Apobec1; designated LDb: LDLR-/-Apobec1-/-), and (ii) mice with the LDb background, who also overexpressed human apoB100 (designated LTp: LDLR-/-Apobec1-/-ERhB+/+). Both LDb and LTp mice had markedly elevated levels of LDL and increased levels of NEFAs (non-esterified fatty acids) compared with C57BL/6 wild-type mice. However, fasting glucose and insulin levels in both animals were not different than those in C57BL/6 wild-type mice. It has been suggested that PAF-AH (platelet-activating factor acetylhydrolase) increases susceptibility to vascular disease. Both LDb and LTp mice had significantly higher PAF-AH mRNA levels compared with C57BL/6 wild-type mice. PAF-AH gene expression was also significantly influenced by age and sex. Interestingly, PAF-AH mRNA levels were significantly higher in both LTp male and female mice than in the LDb mice. This increased PAF-AH gene expression was associated with elevated plasma PAF-AH enzyme activities (LTp>LDb>C57BL/6). Moreover, a greater proportion of PAF-AH activity was associated with the apoB-containing lipoproteins: 29% in LTp and 13% in LDb mice compared with C57BL/6 wild-type animals (6.7%). This may explain why LTp mice developed more atherosclerotic lesions than LDb mice by 8 months of age. In summary, increased plasma NEFAs, PAF-AH mRNA and enzyme activities are associated with accelerated atherogenesis in these animal models.
Pulmonary Vein Isolation for Vagotonic, Adrenergic, and Random Episodes of Paroxysmal Atrial Fibrillation
Authors: ORAL HAKAN , CHUGH AMAN , SCHARF CHRISTOPH , HALL BURR , CHEUNG PETER , VEERAREDDY SRIKAR , DANESHVAR GERALD F. , PELOSI FRANK , MORADY FRED
Published: April 2004

Introduction: Based on the clinical history, paroxysmal atrial fibrillation (PAF) may be classified as vagotonic, adrenergic, or random. It is unclear whether pulmonary vein (PV) isolation is equally effective for these types of PAF.

Methods and Results: Segmental ostial ablation to isolate the PVs was performed in 188 consecutive patients (mean age 53 ± 12 years) with PAF. Based on the clinical history, PAF was classified as random in 136 patients (72%), adrenergic in 30 (16%), and vagotonic in 22 (12%). Three or four PVs were targeted for isolation in all patients, and successful isolation was achieved in 96% of targeted PVs. At 1‐year follow‐up, 69% of patients with random AF, 83% of patients with adrenergic AF, and 50% of patients with vagotonic AF were free from recurrent episodes of AF in the absence of any antiarrhythmic drug therapy. Vagotonic AF was an independent clinical predictor of recurrent AF (P = 0.03).

Conclusion: PV isolation has a lower efficacy in patients with vagotonic PAF than in patients with adrenergic or random episodes of PAF, suggesting that the PVs less often play an important role in vagotonic PAF. (J Cardiovasc Electrophysiol, Vol. 15, pp. 402‐405, April 2004)
Dilated Cardiomyopathy Associated with Dual‐Chamber Pacing in Infants:
Authors: JANOUŠEK JAN , TOMEK VIKTOR , CHALOUPECKÝ VÁCLAV , GEBAUER ROMAN ANTONÍN
Published: April 2004

The etiology of dilated cardiomyopathy associated with congenital complete AV block has not yet been clarified. Two infants with AV block of autoimmune and surgical etiology, respectively, had received a dual‐chamber right ventricular‐based pacemaker and developed dilated cardiomyopathy with severe septal to left ventricular free‐wall dyssynchrony 3.4 (0.9) years later. After 4 weeks of biventricular pacing and spontaneous junctional narrow QRS rhythm, respectively, both children showed significant improvement in left ventricular function along with reverse remodeling. Thus, electromechanical dyssynchrony associated with conventional right‐ventricular‐based DDD pacing may play a significant role in the development of dilated cardiomyopathy in the young. (J Cardiovasc Electrophysiol, Vol. 15, pp. 470‐474, April 2004)
Quality‐of‐Life Effects of Psychosocial Intervention in Patients with Head and Neck Cancer
Authors: Hammerlid Eva , Persson Lars‐Olof , Sullivan Marianne , Westin Thomas
Published: April 2004

METHODS

Two studies of psychosocial interventions in head and neck cancer patients at different stages of their disease were performed. We explored the feasibility and effectiveness of different approaches, offered for the first time to this population. The first study concerned long‐term group psychological therapy for patients with newly diagnosed head and neck cancer. Quality of life was measured longitudinally for 1 year and compared with that of a control group. The second study comprised a short‐term psychoeducational program 1 year after treatment for head and neck cancer. Quality‐of‐life assessments were made repeatedly from diagnosis until 1 month after the intervention. RESULTS: Thirteen patients started the psychological group therapy, and 8 of them completed both the intervention and evaluation procedure. The quality of life of the therapy group improved more than that of a control group in most areas measured during the study year, in particular psychiatric morbidity, social functioning, emotional functioning, and global quality of life. The results indicate benefits from the therapy, although the therapy group scored worse than the control group at diagnosis. Most of the variables representing functioning and symptoms improved after the 1‐week psychoeducational program, especially items reflecting “trouble eating” and “problems enjoying your meals.” Patients' judgments of the intervention quality indicated satisfaction with all separate elements, mostly education, about cancer and the opportunity to socialize with the other guests.

CONCLUSION

These pilot studies suggest that head and neck cancer patients can benefit from different psychosocial interventions. Quality‐of‐life questionnaires were well accepted and sensitive to changes during the studies. Thus our early findings seem promising and would justify confirmation in larger studies. (Otolaryngol Head Neck Surg 1999;120:507‐16.)
Morphologic Changes in the Inner Ear of Chinchilla Laniger after Middle Ear Administration of Gentamicin in a Sustained‐Release Vehicle
Authors: Hoffer Michael E. , Balough Ben J. , Kopke Richard D. , Henderson Jenifer , Decicco Michael , Wester Derin C. , O'Leary Michael J. , Balaban Carey
Published: April 2004

The use of transtympanic gentamicin has become a popular method of treating Meniere's disease; nevertheless, many questions still remain regarding this therapy. Until investigators can control the exact amount of medicine that is administered to the ear and have an understanding of the kinetics of gentamicin, therapy will continue to rely on empirical data. Previously we described the use of a fibrin‐based sustained‐release vehicle impregnated with gentamicin in the middle ears of chinchillas. With this model a kinetics curve of gentamicin was defined. The inner ears of these animals were submitted for immunohistochemical and histologic analysis. We discuss the ultrastructural changes seen and correlate this to our kinetics data. We also examine measurement of hair cell damage with heat shock protein levels. By better understanding the actions of gentamicin in this animal model, we hope to facilitate safer use of intratympanic medicines in our patient population.
Long‐Term Growth of a Vascularized Auricular Perichondrocutaneous Flap in Laryngotracheal Reconstruction
Authors: Hoff Paul T. , Esclamado Ramon M.
Published: April 2004

This study addresses the potential for ongoing cartilage proliferation after repair of laryngotracheal stenosis with vascularized perichondrium. We randomly assigned 32 New Zealand white rabbits to 1 of 3 groups: group 1 (early cartilage growth, n = 10), group 2 (long‐term cartilage growth after pedicle ligation, n = 11), and group 3 (long‐term cartilage growth without pedicle ligation, n = 11). Bilateral auricular perichondrocutaneous flaps were elevated and transposed into full‐thickness anterior tracheal wall or anterior cricothyroid membrane defects. Six weeks after elevation of the flap, animals were randomly assigned to undergo ligation of either the right or left vascular pedicle (group 2), with the contralateral auricular flap used as a matched control (group 3). Neochondrogenesis was present at 6 weeks in group 1 (0.74 ± 0.14 mm, n = 12 ears). Cartilage thickness did not differ between groups 2 and 3 one year after ligation of the vascular pedicle: group 2 (0.48 ± 0.24 mm, n = 18) versus group 3 (0.42 ± 0.12 mm); P > 0.05. We conclude that in the rabbit model, chondrogenesis did not appear to be ongoing and did not result in late stenosis of the reconstructed airway. Furthermore, delayed ligation of the vascular pedicle neither inhibited nor stimulated cartilage proliferation.
Who is Doing What to Whom? Young Infants' Developing Sense of Social Causality in Animated Displays
Authors: Rochat Philippe , Striano Tricia , Morgan Rachel
Published: April 2004

In two different experiments a visual habituation/dishabituation procedure was used to test groups of 3–10-month-old infants for their ability to discriminate the role reversal of two abstract figures (discs of different colors) chasing each other on a computer screen. Results of the first experiment point to a reliable age effect. Only 8–10-month-old infants tended to dishabituate to a role reversal between chaser and chasee. A second experiment shows that in dishabituating to the role reversal, 8–10-month-olds do base this discrimination on relational information between the two discs and not merely on the contrast between their respective vitality or discrete dynamic. By the age of 8–10 months, infants demonstrate sensitivity to information specifying what one disc does to the other, at a distance. These findings point to important changes in perceptual-cognitive development and are discussed in the context of a well described key transition in social-cognitive development occurring at around 9 months of age.
A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by
Nicotiana benthamiana
Authors: Yang Shu-Jun , Carter Shelly A. , Cole Anthony B. , Cheng Ning-Hui , Nelson Richard S.
Published: April 2004

Nicotiana benthamiana
often displays more intense symptoms after infection by RNA viruses than do other
Nicotiana
species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in
N
.
benthamiana
antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in
N
.
benthamiana
. One
RdRP
was similar in sequence to
SDE1
/
SGS2
required for maintenance of transgene silencing, whereas the second, named
NbRdRP1m
, was >90% identical in sequence to the salicylic acid (SA)-inducible
RdRP
from
Nicotiana tabacum
required for defense against viruses.
NbRdRP1m
expression was induced by SA treatment or challenge with
Tobacco mosaic virus
, but the gene and transcript sequences differed from those of other SA-inducible
RdRP
s in that they contained a 72-nt insert with tandem in-frame stop codons in the 5′ portion of the ORF.
N
.
benthamiana
plants transformed with an SA-inducible
RdRP
gene from
Medicago truncatula
were more resistant to infection by
Tobacco mosaic virus
,
Turnip vein-clearing virus
, and
Sunn hemp mosaic virus
(members of
Tobamovirus
genus), but not to
Cucumber mosaic virus
and
Potato virus X
(members of different genera than the tobamoviruses). Our results indicate that
N
.
benthamiana
lacks an active SA- and virus-inducible RdRP and thus is hypersusceptible to viruses normally limited in their accumulation by this RdRP. These findings are significant for those studying virus-induced gene silencing, the hypersensitive response and systemic acquired resistance.
Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4
+
T lymphocytes
Authors: Marañón Concepción , Desoutter Jean-François , Hoeffel Guillaume , Cohen William , Hanau Daniel , Hosmalin Anne
Published: April 2004

A better understanding of the antigen presentation pathways that lead to CD8
+
T cell recognition of HIV epitopes
in vivo
is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4
+
T lymphocytes by dendritic cells to CD8
+
T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4
+
T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4
+
T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFNα immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.
Asymmetric catalysis: An enabling science
Authors: Trost Barry M.
Published: April 2004

Chirality of organic molecules plays an enormous role in areas ranging from medicine to material science, yet the synthesis of such entities in one enantiomeric form is one of the most difficult challenges. The advances being made stem from the convergence of a broader understanding of theory and how structure begets function, the developments in the interface between organic and inorganic chemistry and, most notably, the organic chemistry of the transition metals, and the continuing advancements in the tools to help define structure, especially in solution. General themes for designing catalysts to effect asymmetric induction are helping to make this strategy more useful, in general, with the resultant effect of a marked enhancement of synthetic efficiency.
Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center
Authors: Baxter Richard H. G. , Ponomarenko Nina , Šrajer Vukica , Pahl Reinhard , Moffat Keith , Norris James R.
Published: April 2004

Light-induced structural changes in the bacterial reaction center were studied by a time-resolved crystallographic experiment. Crystals of protein from
Blastochloris viridis
(formerly
Rhodopseudomonas viridis
) were reconstituted with ubiquinone and analyzed by monochromatic and Laue diffraction, in the dark and 3 ms after illuminating the crystal with a pulsed laser (630 nm, 3 mJ/pulse, 7 ns duration). Refinement of monochromatic data shows that ubiquinone binds only in the “proximal” Q
B
binding site. No significant structural difference was observed between the light and dark datasets; in particular, no quinone motion was detected. This result may be reconciled with previous studies by postulating equilibration of the “distal” and “proximal” binding sites upon extended dark adaption, and in which movement of ubiquinone is not the conformational gate for the first electron transfer between Q
A
and Q
B
.
Zr[bis(salicylidene)ethylenediaminato]-mediated Baeyer–Villiger oxidation: Stereospecific synthesis of abnormal and normal lactones
Authors: Watanabe Akira , Uchida Tatsuya , Irie Ryo , Katsuki Tsutomu
Published: April 2004

Baeyer–Villiger oxidation of racemic bicyclic cyclobutanones with Zr[bis(salicylidene)ethylenediaminato] (salen) complex 1 as catalyst in the presence of a urea-hydrogen peroxide adduct was found to proceed enantiospecifically. The enantiotopos selection in the oxidation was governed primarily by the Zr(salen) catalyst, although migratory aptitude (methine > methylene > methyl) in Baeyer–Villiger oxidation affected the selection to a varied extent, depending on the substrate structures; one enantiomer of cyclobutanones gave exclusively a normal lactone expected from the migratory aptitude, and the other enantiomer gave an abnormal lactone preferentially, the formation of which is counter to the migratory aptitude. Furthermore, the rates of abnormal lactone formation were found to be faster than those of normal lactone formation in most of the oxidations examined. For example, the enantiomer of racemic bicyclo[3.2.0]heptan-6-one giving an abnormal lactone reacted 2.2 times faster than the other enantiomer giving a normal lactone. To our knowledge, this example of chemocatalytic Baeyer–Villiger oxidation giving an abnormal lactone in preference to a normal lactone has been previously unreported. This unusual behavior is likely to be attributable to strict control of stereoelectronic demand in Baeyer–Villiger oxidation and chiral recognition by complex 1.
New biphenol-based, fine-tunable monodentate phosphoramidite ligands for catalytic asymmetric transformations
Authors: Hua Zihao , Vassar Victor C. , Choi Hojae , Ojima Iwao
Published: April 2004

Monodentate phosphoramidite ligands have been developed based on enantiopure 6,6′-dimethylbiphenols with axial chirality. These chiral ligands are easy to prepare and flexible for modifications. The fine-tuning capability of these ligands plays a significant role in achieving high enantioselectivity in the asymmetric hydroformylation of allyl cyanide and the conjugate addition of diethylzinc to cycloalkenones.
Computational predictions of stereochemistry in asymmetric thiazolium- and triazolium-catalyzed benzoin condensations
Authors: Dudding Travis , Houk Kendall N.
Published: April 2004

The catalytic asymmetric thiazolium- and triazolium-catalyzed benzoin condensations of aldehydes and ketones were studied with computational methods. Transition-state geometries were optimized by using Morokuma's IMOMO [integrated MO (molecular orbital) + MO method] variation of ONIOM (
n
-layered integrated molecular orbital method) with a combination of B3LYP/6–31G(d) and AM1 levels of theory, and final transition-state energies were computed with single-point B3LYP/6–31G(d) calculations. Correlations between experiment and theory were found, and the origins of stereoselection were identified. Thiazolium catalysts were predicted to be less selective then triazolium catalysts, a trend also found experimentally.
An efficient and general route to reduced polypropionates via Zr-catalyzed asymmetric C—C bond formation
Authors: Negishi Ei-ichi , Tan Ze , Liang Bo , Novak Tibor
Published: April 2004

An efficient and general method for the synthesis of reduced polypropionates has been developed through the application of asymmetric carboalumination of alkenes catalyzed by dichlorobis(1-neomenthylindenyl)zirconium [(NMI)
2
ZrCl
2
]. In this investigation, attention has been focused on those reduced polypropionates that are α-monoheterofunctional and either ω-ethyl or ω-
n
-propyl. The reaction of 3-buten-1-ol with triethylaluminum (Et
3
Al) or tripropylaluminum (
n
Pr
3
Al) in the presence of (NMI)
2
ZrCl
2
and isobutylaluminoxane gave, after protonolysis, (
R
)-3-methyl-1-pentanol as well as (
R
)- and (
S
)-3-methyl-1-hexanols in 88–92% yield in 90–92% enantiomeric excess in one step. These 3-monomethyl-1-alkanols were then converted to two stereoisomers each of 2,4-dimethyl-1-hexanols and 2,4-dimethyl-1-heptanols via methylalumination catalyzed by (NMI)
2
ZrCl
2
and methylaluminoxane followed by oxidation with O
2
. The four-step (or three-isolation-step) protocol provided
syn
-2,4-dimethyl-1-alkanols of ≥98% stereoisomeric purity in ≈50% overall yields, whereas (2
S
,4
R
)-2,4-dimethyl-1-hexanol of comparable purity was obtained in 40% overall yield. Commercial availability of (
S
)-2-methyl-1-butanol as a relatively inexpensive material suggested its use in the synthesis of (2
S
,4
S
)- and (2
R
,4
S
)-2,4-dimethyl-1-hexanols via a three-step protocol consisting of (
i
) iodination, (
ii
) zincation followed by Pd-catalyzed vinylation, and (
iii
) Zr-catalyzed methylalumination followed by oxidation with O
2
. This three-step protocol is iterative and applicable to the synthesis of reduced polypropionates containing three or more branching methyl groups, rendering this method for the synthesis of reduced polypropionates generally applicable. Its synthetic utility has been demonstrated by preparing the side chain of zaragozic acid A and the C11–C20 fragment of antibiotics TMC-151 A–F.
A pathway for phosphatidylcholine biosynthesis inPlasmodium falciparuminvolving phosphoethanolamine methylation
Authors: Pessi Gabriella , Kociubinski Guillermo , Mamoun Choukri Ben
Published: April 2004

Plasmodium falciparumis the causative agent of the most severe form of human malaria. The rapid multiplication of the parasite within human erythrocytes requires an active production of new membranes. Phosphatidylcholine is the most abundant phospholipid inPlasmodiummembranes, and the pathways leading to its synthesis are attractive targets for chemotherapy. In addition to its synthesis from choline, phosphatidylcholine is synthesized from serine via an unknown pathway. Serine, which is actively transported byPlasmodiumfrom human serum and readily available in the parasite, is subsequently converted into phosphoethanolamine. Here, we describe inP. falciparuma plant-likeS-adenosyl-l-methionine-dependent three-step methylation reaction that converts phosphoethanolamine into phosphocholine, a precursor for the synthesis of phosphatidylcholine. We have identified the gene,PfPMT, encoding this activity and shown that its product is an unusual phosphoethanolamine methyltransferase with no human homologs.P. falciparumphosphoethanolamine methyltransferase (Pfpmt) is a monopartite enzyme with a single catalytic domain that is responsible for the three-step methylation reaction. Interestingly, Pfpmt activity is inhibited by its product phosphocholine and by the phosphocholine analog, miltefosine. We show that miltefosine can also inhibit parasite proliferation within human erythrocytes. The importance of this enzyme inP. falciparummembrane biogenesis makes it a potential target for malaria chemotherapy.