bluuebunny/crossref_metadata_2025 huggingface dataset:
Dopamine in the ink defence system of Sepia officinalis: biosynthesis, vesicular compartmentation in mature ink gland cells, nitric oxide (NO)/cGMP-induced depletion and fate in secreted ink
Authors: FIORE Gabriella , POLI Annarita , Di COSMO Anna , d'ISCHIA Marco , PALUMBO Anna
Published: April 2004

The biosynthesis, localization and fate of catecholamines in the ink gland of the cuttlefish Sepia officinalis were investigated by combined biochemical and immunohistocytochemical methodologies. HPLC analysis of crude ink gland extracts indicated the presence of dopa (2.18±0.82 nmol/mg of protein) and DA (dopamine, 0.06±0.02 nmol/mg of protein), but no detectable noradrenaline or adrenaline. DA was shown to derive from l-tyrosine, according to experiments performed by incubating intact ink glands with [l-14C]tyrosine. The biosynthetic process involves a tyrosine hydroxylase and a dopa decarboxylase pathway and is independent of tyrosinase. The tyrosine hydroxylase activity was detected under conditions of tyrosinase suppression in the cytosolic fraction, but not in the melanosomal fraction, of ink gland extracts, and the presence of the enzyme was confirmed by Western-blot analysis. Dopa and DA were found to be released from the ink glands by processes controlled through the NMDA-nitric oxide-cGMP (where NMDA stands for N-methyl-d-aspartate) signalling pathway, as apparent from incubation experiments performed with [l-14C]tyrosine in the presence of NMDA, diethylamine NONOate (diethylamine diazeniumdiolate), a nitric oxide donor, 8-bromo-cGMP or a guanylyl cyclase inhibitor. Immunohistochemical results coupled with electron microscopy indicated that DA was concentrated in vesicles specifically localized in the mature melanin-producing cells of the ink gland proximal to the lumen and separated from the melanin-containing melanosomes. NMDA receptor stimulation or exposure to an NO donor caused a marked loss of DA immunoreactivity in mature cells, consistent with a release process. In the lumen of the ink gland, where mature exhausted cells pour their contents, DA immunoreactivity was found to be associated with the melanin granules, due apparently to physical adsorption. Overall, these results point to DA as a marker of cell maturation in Sepia ink gland subject to release by the NO/cGMP signalling pathway, and disclose apparently overlooked DA–melanin interactions in secreted ink of possible relevance to the defence mechanism.
Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles
Authors: NORI Alessandra , BORTOLOSO Elena , FRASSON Federica , VALLE Giorgia , VOLPE Pompeo
Published: April 2004

CS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation–contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sar1 and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79→Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis-medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeting to either TC or developing SR. Thus active budding from ER-exit sites appears to be involved in CS targeting and routing, but these processes are insensitive to modification of intracellular vesicle trafficking and Golgi complex disruption caused by the mutant ARF1-N126I. It also appears that CS routing from ER to SR does not involve classical secretory pathways through ER–Golgi intermediate compartments, cis-medial Golgi and trans-Golgi network.
Voluntary breath-holding in the morning and in the evening
Authors: BOSCO Gerardo , IONADI Alessandro , DATA Piergiorgio G. , MORTOLA Jacopo P.
Published: April 2004

The aim of the present study was to determine whether or not voluntary breath-holding time (BHT) changes with the time of the day. BHT with airways closed at end-expiration was measured in six male subjects in the sitting position during the morning (08.00–12.00 hours, on days 1, 6, 7 and 8) and evening (20.00–24.00 hours, on days 2 and 4). BHT increased with the number of days of testing and, at day 8, the morning values averaged 160% of those on day 1. Also, ΔPACO2 [the difference between end-tidal partial pressure of CO2 (PCO2) and alveolar PCO2 (PACO2) at the breaking point] increased in proportion to BHT. Hence the BHT/ΔPACO2 ratio remained nearly constant. Voluntary hyperventilation prolonged BHT and increased ΔPACO2. Conversely, in hypoxia (13% O2 for 1–2 h), BHT and ΔPACO2 were reduced proportionally. During the evening sessions, most of the BHT/ΔPACO2 ratios in normoxia, hypoxia or after hyperventilation were higher than the corresponding morning values, with the group difference reaching statistical significance for the measurements in normoxia and hypoxia. In conclusion, voluntary BHT varies in both duration and its relationship with ΔPACO2 between the morning and evening hours. The results should also imply that, with an interruption of breathing, changes in alveolar and arterial gases are not the same at different times of the day.
Plasma urocortin in human systolic heart failure
Authors: NG Leong L. , LOKE Ian W. , O'BRIEN Russell J. , SQUIRE Iain B. , DAVIES Joan E.
Published: April 2004

Urocortin (UCN), a member of the corticotrophin-releasing factor family, is expressed in heart, brain and gut. UCN has potent cardiostimulatory, cardioprotective, vasodilator and diuretic/natriuretic effects, and cardiac UCN expression is increased in heart failure (HF). In the present study, we investigated plasma levels of UCN in 119 patients with HF and 212 age- and gender-matched controls to clarify its relationship with gender and disease severity. UCN was elevated in HF [normal males, 19.5 (3.9–68.8) pmol/l and HF males, 50.2 (6.9–108.2) pmol/l, P<0.0005; normal females, 14.2 (3.9–53.5) pmol/l and HF females, 21.8 (3.9–112.5) pmol/l, P<0.001; values are medians (range)]. The relative increase was greater in males than females (P<0.03). UCN fell with increasing age, especially in HF patients (rs=-0.56, P<0.0005) and with increasing New York Heart Association (NYHA) class (rs=-0.55, P<0.0005). The fall in UCN levels with increasing NYHA class was reinforced by a significant correlation between UCN and ejection fraction (rs=0.45, P<0.0005) in HF patients. Although receiver operating characteristic (ROC) curves for diagnosis of all HF cases yielded an area under the curve (AUC) of 0.76, ROC AUCs for patients with early HF (NYHA class I and II) were better (0.91). ROC AUCs for logistic models incorporating N-terminal probrain natriuretic peptide (N-BNP) and UCN were better than either peptide alone. In conclusion, plasma UCN is elevated in HF, especially in its early stages. Its decline with increasing HF severity may expedite disease progression due to diminished cardioprotective/anti-inflammatory effects. UCN measurement may also complement N-BNP in the diagnosis of early HF.
Second trimester Doppler ultrasound screening of the uterine arteries differentiates between subsequent normal and poor outcomes of hypertensive pregnancy: two different pathophysiological entities?
Authors: AARDEMA M. W. , SARO M. C. S. , LANDER M. , M. DE WOLF B. T. H. , OOSTERHOF H. , AARNOUDSE J. G.
Published: April 2004

The ‘classical’ concept that pregnancy-induced hypertension (PIH) and pre-eclampsia (PE) primarily originate from defective placentation in early pregnancy has been challenged recently. There is growing evidence that other factors, including maternal predisposing conditions, also play a significant role in the pathophysiology of PIH and PE. The aim of the present study was to test the hypothesis that PIH and PE with an early onset and poor pregnancy outcome is associated with defective placentation, e.g. inadequate spiral artery dilatation and subsequent reduced uteroplacental perfusion, whereas PIH and PE with normal pregnancy outcome is not. Using Doppler ultrasound, we measured the uterine artery pulsatility index (PI) in a population of 531 nulliparous women in the 22nd week of gestation. Uterine artery PI was used as an index of resistance to blood flow in the uteroplacental circulation. Outcome measures were PIH/PE with or without poor pregnancy outcome, preterm birth and intra-uterine growth restriction (IUGR). The results revealed a striking difference between PI values for PIH/PE with and without poor pregnancy outcome. Uterine artery PI in the 22nd week was increased significantly in pregnancies which developed early-onset (before 35 weeks) PIH/PE with a poor pregnancy outcome. In contrast, uterine artery PI values were normal in women who developed PIH/PE, but had a good pregnancy outcome. There was a significant correlation between 22nd week uterine artery PI and subsequent preterm birth or IUGR. Our results indicate that only PIH/PE with poor pregnancy outcome is associated with defective placentation, whereas PIH/PE with good outcome is not. These findings support the concept of heterogeneous causes of hypertensive disorders of pregnancy.
First Human Chronic Experience with Cardiac Contractility Modulation by Nonexcitatory Electrical Currents for Treating Systolic Heart Failure:
Authors: PAPPONE CARLO , AUGELLO GIUSEPPE , ROSANIO SALVATORE , VICEDOMINI GABRIELE , SANTINELLI VINCENZO , ROMANO MASSIMO , AGRICOLA EUSTACHIO , MAGGI FRANCESCO , BUCHMAYR GERHARD , MORETTI GIOVANNI , MIKA YUVAL , BEN‐HAIM SHLOMO A. , WOLZT MICHAEL , STIX GUENTER , SCHMIDINGER HERWIG
Published: April 2004

Introduction: Conventional electrical therapies for heart failure (HF) encompass defibrillation and ventricular resynchronization for patients at high risk for lethal arrhythmias and/or with inhomogeneous ventricular contraction. Cardiac contractility modulation (CCM) by means of nonexcitatory electrical currents delivered during the action potential plateau has been shown to acutely enhance systolic function in humans with HF. The aim of this multicenter study was to assess the chronic safety and preliminary efficacy of an implantable device delivering this novel form of electrical therapy.

Methods and Results: Thirteen patients with drug‐resistant HF (New York Heart Association [NYHA] class III) were consecutively implanted with a device (OPTIMIZER™ II) delivering CCM biphasic square‐wave pulses (20 ms, 5.8–7.7 V, 30 ms after detection of local activation) through two right ventricular leads screwed into the right aspect of the interventricular septum. CCM signals were delivered 3 hours daily over 8 weeks (3‐hour phase) and 7 hours daily over the next 24 weeks (7‐hour phase). Safety and feasibility of this novel therapy were regarded as primary endpoints. Preliminary clinical efficacy, ‐as expressed by changes in ejection fraction (EF), NYHA class, 6‐minute walking test (6‐MWT), peak O2 uptake (peak VO2), and Minnesota Living with HF Questionnaire (MLWHFQ), was assessed at baseline and at the end of each phase. At the end of follow‐up (8.8 ± 0.2 months), all patients were alive, without heart transplantation or need for left ventricular assist device. Serial 24‐hour Holter analysis revealed no proarrhythmic effect. No devices malfunctioned or failed for any reason other than end‐of‐battery life. Throughout the two study phases, EF improved from 22.7 ± 7% to 28.7 ± 7% and 37 ± 13% (P = 0.004), 6‐MWT from 418 ± 99 m to 477 ± 96 m and 510 ± 107 m (P = 0.002), MLWHFQ from 36 ± 21 to 18 ± 12 and 7 ± 6 (P = 0.002), peak VO2 from 13.7 ± 1.1 to 14.9 ± 1.9 to 16.2 ± 2.4 (P = 0.037), and NYHA class from 3 to 1.8 ± 0.4 to 1.5 ± 0.7 (P < 0.001).

Conclusion: CCM therapy appears to be safe and feasible. Proarrhythmic effects of this novel therapy seem unlikely. Preliminary data indicate that CCM gradually and significantly improves systolic performance, symptoms, and functional status. CCM therapy for 7 hours per day is associated with greater dispersion near the mean, emphasizing the need to individually tailor CCM delivery duration. The technique appears to be attractive as an additive treatment for severe HF. Controlled randomized studies are needed to validate this novel concept. (J Cardiovasc Electrophysiol, Vol. 15, pp. 418‐427, April 2004)
Ultrastructural Aspects of the Microvasculature of the Cochlea: The Internal Spiral Network
Authors: Penha Rui , O'Neill Maria Assunção , O'Neill João Goyri , Pina José António Esperança
Published: April 2004

We performed a study of the microvasculature of the cochlea with scanning electron microscopy on corrosion casts in the guinea pig. This study was performed in 140 cochleas from healthy adult guinea pigs. Different microvascular techniques were used, including injection‐microdissection in 53 cases, injection‐diaphanization in 27 cases, a histologic technique in 34 cases, injection‐microdissection‐diaphanization in 32 cases, and scanning electron microscopy on corrosion casts in 14 cases. The internal radiating arterioles branch off as collateral branches of the spiral modiolar artery, as first‐order collateral branches. We detail the morphology, caliber, trajectory, and collateral branches. Among their collateral branches, are third‐order arterioles, the arterioles to the spiral ganglia, and the arterioles of the tympanic lip. The arterioles of the tympanic lip form, through their anastomosis, a rich capillary network at the edge of the spiral lamina, called the internal spiral network. We emphasize the glomeruli of Schwalbe, which arise near the scala tympani as third‐order arterioles with a medium caliber of 14 μm. The upper glomeruli, situated in the bony wall, and the lower glomeruli, situated in the width of the osseous spiral lamina, form vascular loops made of anastomosed capillaries with a caliber of less than 10 μm. These loops play an important role as efficient devices, or “relay stations,” for regulation of cochlear blood flow. The comparison of results obtained with each technique gave the perspective of the cochlear microvasculature with great accuracy.
Treatment of the Patient with Upper Airway Obstruction Caused by Cancer of the Larynx
Authors: Bradley Patrick J.
Published: April 2004

The treatment of a patient with imminent airway obstruction caused by a malignant tumor of the larynx is an uncommon clinical problem. These cases need to be evaluated, diagnosed, and managed with care, skill, speed, and above all, appropriateness of intervention. Three methods are available to control the airway: tracheostomy, emergency laryngectomy, and controlled tracheal intubation with or without tumor debulking. Two groups of patients had their airways managed either by tracheostomy and delayed elective surgery or by emergency laryngectomy. There was no survival advantage between the groups, and no increased risk of stomal recurrence was demonstrated. If time permits, the patient is considered suitable, and adequate anesthetic and surgical instrumentation is available, it is currently recommended that the obstructing laryngeal tumor be debulked by cold‐steel or, preferably, CO2 laser and that the emergency situation be stabilized and the definitive treatment of the patient be converted to an elective procedure without the need to create a tracheostomy.
Occult Cervical Metastases: Immunoperoxidase Analysis of the Pathologically Negative Neck
Authors: Enepekides Danny J. , Sultanem Khaleil , Nguyen Cam , Shenouda George , Black Martin J. , Rochon Louise
Published: April 2004

OBJECTIVES

The purpose of this study is to better estimate the true incidence of occult regional metastases associated with stage I and II squamous cell carcinoma of the oral cavity. The clinical and prognostic significance of micrometastatic disease discovered by cytokeratin immunoperoxidase reactivity in the previously pathologically N0 neck is also evaluated.

METHODS

Forty patients treated between 1985 and 1996 with T1 or T2 squamous cell carcinoma of the lip and oral cavity were studied. All had primary surgical treatment including functional neck dissection. No metastases were demonstrated on hematoxylin and eosin microscopy. All specimens were reexamined with immunoperoxidase staining for cytokeratin.

RESULTS

Five percent of patients had micrometastatic disease. Retrospective analysis of patients with a minimum follow‐up of 2 years has failed to show a statistically significant association between a positive cytokeratin analysis and poor locoregional control or overall survival.

CONCLUSIONS

Results suggest that the true incidence of occult metastases with carcinoma of the oral cavity is significantly higher than previously documented. However, the prognostic significance of these findings remains unclear.
Evidence of Oxygen Free Radical Damage in Human Otitis Media
Authors: Takoudes Thomas G. , Haddad Joseph
Published: April 2004

Recent work with a guinea pig model of otitis media has demonstrated evidence of oxygen free radical damage to the middle ear mucosa. However, the relevance of an animal model to human disease is uncertain. Accordingly, the following pilot study was conducted to examine human middle ear fluid for lipid hydroperoxides as evidence of free radical damage. Thirty‐five specimens of middle ear fluid from children with chronic otitis media were collected and described as mucoid (n = 19), purulent (n = 10), or serous (n = 6); specimens were weighed and analyzed for lipid hydroperoxide content. The results demonstrated the presence of lipid hydroperoxide in all 3 types of middle ear fluid. Additionally, there was a statistically significant elevation of total lipid hydroperoxide content in mucoid effusions compared with serous effusions, as well as a significant elevation of lipid hydroperoxide divided by weight of purulent effusions compared with serous effusions. This is the first study to document free radical damage in human otitis media.
High‐Risk Human Papillomavirus Types and Squamous Cell Carcinoma in Patients with Respiratory Papillomas
Authors: Moore Charles E. , Wiatrak Brian J. , Mcclatchey Kenneth D. , Koopmann Charles F. , Thomas Giovanna R. , Bradford Carol R. , Carey Thomas E.
Published: April 2004

Respiratory papillomas (RPs) are benign, virally induced tumors of the larynx and respiratory epithelium that may obstruct the airway and tend to recur frequently. RPs are thought to be the result of infection with the human papillomaviruses (HPVs) types 6 and 11. We surveyed archival RP specimens to determine whether there were correlations of HPV type with patient characteristics or clinical course. Paraffin‐embedded papilloma specimens of 45 different patients were analyzed. We assessed HPV types using the polymerase chain reaction with E6 consensus primers, hybrid capture assays (high or low risk), and dot blot hybridization of generic E6 PCR products with E6 type‐specific oligonucleotide probes. The presence and type of HPV were correlated with patient data from a retrospective chart review. We found that RPs may have either low‐ or high‐risk HPV types and some contain multiple HPV types. Respiratory infection with high‐risk HPV apparently introduces a long‐term risk of squamous cell carcinoma development, even in the absence of conventional cofactors. Low‐risk HPV infection may also act in association with these cofactors to promote carcinogenesis. Our data also show a racial imbalance in RP that may indicate a difference in genetic resistance and/or susceptibility to HPV infection and the development of RP.
Gearbox (China) Ltd.: Will the Company's ERP System Support its Ambitious Growth Strategy?
Authors: Reimers Kai
Published: April 2004

This case describes the experience of a wholly foreign-owned manufacturing company in Tianjin/China regarding the use of its ERP system in its main functional departments, purchasing, production planning, sales/distribution, and finance. The company is part of a group which is a global leader in the manufacturing and distribution of mechanical devices, called gearboxes, that are needed to drive a wide range of facilities such as escalators and baggage conveyor belts in airports. It has entered China in 1995 and the Tianjin manufacturing facility has soon become a hub for the Asian market. The main challenge confronting the management team is to support the breakneck growth rate of this young company. The company's ERP system plays a crucial role in this task. However, it seems that middle managers are frequently hitting an invisible wall when trying to expand the use of the ERP system in order to cope with ever-increasing workloads and coordination tasks. This case serves to illustrate cultural issues implicated in the use of an enterprise wide information system in a medium size company operating in an emerging market economy. In addition, issues of operations management, global management, and organizational behaviour are addressed.
Working in Partnership for Regeneration—The Effect of Organisational Norms on Community Groups
Authors: O'Malley Lisa
Published: April 2004

This paper draws on theories of new institutionalism to examine the consequences of partnership working for the community sector. Three normative practices which are associated with partnership working—representativeness; consensus-based decisionmaking; and bureaucratic styles of working—are examined in relation to the experiences of two UK case-study localities in receipt of Single Regeneration Budget funding. The findings reflect a complex interaction between acceptance and noncompliance with these norms by community organisations which leads to a variety of outcomes for individual community representatives and for relations between community groups and regeneration partnership boards. In conclusion, it is argued that protecting the independence of local organisations can allow for opposition to partnership-based decisions but that this risks undermining holistic or strategic approaches to social problems. The actions of some community organisations in these two localities suggests that, although some aspects of partnership working are increasingly institutionalised within community sectors, organisations remain alert to opportunities to subvert these norms to further their own aspirations.
A Dual Neural Network for Solving Entropy-Maximising Models
Authors: Leung Yee , Gao Xing-Bao , Chen Kai-Zhou
Published: April 2004

The entropy-maximixing model has been applied with varying degrees of success in the analysis and planning of origin – destination types of spatial interaction. Although theoretical underpinnings and solution methods have been developed over the years, there are still outstanding problems that need to be thoroughly investigated. From the practical point of view, solving this model directly and in real time has high theoretical and pragmatic value. In this paper we propose a neural network for solving the dual problem of this model in real time. The size of the proposed network is very small and its structure is very simple, so it can be implemented in hardware. From the theoretical perspective, we solve the seldom investigated issue of convergence to the optimal solution of the entropy-maximising model. We strictly prove that the proposed dual neural network is Lyapunov stable and that each of its trajectories can converge asymptotically to an exact solution of the dual problem. The validity and transient behaviour of the proposed neural network are demonstrated by numerical examples. It is also demonstrated that the proposed network approach renders for the first time a tight integration of an entropy-maximising model and a neural network, and offers a general representation and solution to a large variety of entropy-maximising models.
Single-molecule assay reveals strand switching and enhanced processivity of UvrD
Authors: Dessinges Marie-Noëlle , Lionnet Timothée , Xi Xu Guang , Bensimon David , Croquette Vincent
Published: April 2004

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3′-5′ on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.
Network motifs in integrated cellular networks of transcription–regulation and protein–protein interaction
Authors: Yeger-Lotem Esti , Sattath Shmuel , Kashtan Nadav , Itzkovitz Shalev , Milo Ron , Pinter Ron Y. , Alon Uri , Margalit Hanah
Published: April 2004

Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription–regulation or protein–protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription–regulation and protein–protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein–protein interactions and transcription regulation in
Saccharomyces cerevisiae
. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.
Enantioselective direct aldol reactions catalyzed byl-prolinamide derivatives
Authors: Tang Zhuo , Jiang Fan , Cui Xin , Gong Liu-Zhu , Mi Ai-Qiao , Jiang Yao-Zhong , Wu Yun-Dong
Published: April 2004

l-Prolinamides 2, prepared froml-proline and simple aliphatic and aromatic amines, have been found to be active catalysts for the direct aldol reaction of 4-nitrobenzaldehyde with neat acetone at room temperature. They give moderate enantioselectivities of up to 46% enantiomeric excess (ee). The enantioselectivity increases as the amide N—H becomes a better hydrogen bond donor.l-Prolinamides 3, derived from the reaction ofl-proline with α,β-hydroxyamines such that there is a terminal hydroxyl group, show more efficient catalysis and higher enantioselectivities. In particular, catalyst 3h, prepared froml-proline and (1S,2S)-diphenyl-2-aminoethanol, exhibits high enantioselectivities of up to 93% ee for aromatic aldehydes and up to >99% ee for aliphatic aldehydes under –25°C. Model reactions of benzaldehyde with three enamines derived from the condensation of prolinamides with acetone have been studied by quantum mechanics calculations. The calculations reveal that the amide N—H and the terminal hydroxyl groups form hydrogen bonds with the benzaldehyde substrate. These hydrogen bonds reduce the activation energy and cause high enantioselectivity. Our results suggest a new strategy in the design of new organic catalysts for direct asymmetric aldol reactions and related transformations.
Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: An anthrax toxin receptor
Authors: Lacy D. Borden , Wigelsworth Darran J. , Scobie Heather M. , Young John A. T. , Collier R. John
Published: April 2004

Anthrax toxin is released from
Bacillus anthracis
as three monomeric proteins, which assemble into toxic complexes at the surface of receptor-bearing host cells. One of the proteins, protective antigen (PA), binds to receptors and orchestrates the delivery of the other two (the lethal and edema factors) into the cytosol. PA has been shown to bind to two cellular receptors: anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (CMG2). Both are type 1 membrane proteins that include an ≈200-aa extracellular von Willebrand factor A (VWA) domain with a metal ion-dependent adhesion site (MIDAS) motif. The anthrax toxin receptor/tumor endothelial marker 8 and CMG2 VWA domains share ≈60% amino acid identity and bind PA directly in a metal-dependent manner. Here, we report the crystal structure of the CMG2 VWA domain, with and without its intramolecular disulfide bond, to 1.5 and 1.8 Å, respectively. Both structures contain a carboxylate ligand-mimetic bound at the MIDAS and appear as open conformations when compared with the VWA domains from α-integrins. The CMG2 structures provide a template to begin probing the high-affinity CMG2–PA interaction (200 pM) and may facilitate understanding of toxin assembly/internalization and the development of new anthrax treatments. The structural data also allow molecular interpretation of known CMG2 VWA domain mutations linked to the genetic disorders, juvenile hyaline fibromatosis, and infantile systemic hyalinosis.
Switch from intracellular to intercellular invasion during water stress-tolerant legume nodulation
Authors: Goormachtig Sofie , Capoen Ward , James Euan K. , Holsters Marcelle
Published: April 2004

Rhizobia colonize their legume hosts by different modes of entry while initiating symbiotic nitrogen fixation. Most legumes are invaded via growing root hairs by the root hair-curl mechanism, which involves epidermal cell responses. However, invasion of a number of tropical legumes happens through fissures at lateral root bases by cortical, intercellular crack entry. In the semiaquatic
Sesbania rostrata
, the bacteria entered via root hair curls under nonflooding conditions. Upon flooding, root hair growth was prevented, invasion on accessible root hairs was inhibited, and intercellular invasion was recruited. The plant hormone ethylene was involved in these processes. The occurrence of both invasion pathways on the same host plant enabled a comparison to be made of the structural requirements for the perception of nodulation factors, which were more stringent for the epidermal root hair invasion than for the cortical intercellular invasion at lateral root bases.
Distinct dosage requirements for the maintenance of long and short telomeres in
mTert
heterozygous mice
Authors: Erdmann Natalie , Liu Yie , Harrington Lea
Published: April 2004

Telomerase is a ribonucleoprotein containing an essential telomerase RNA template and telomerase reverse transcriptase (TERT) that maintains telomeres. The dosage requirements for mammalian TERT in telomere length homeostasis are not known, but are of importance in cellular senescence, stem cell renewal, and cancer. Here, we characterize telomere maintenance and function upon successive breeding of mice deficient in
mTert
. These studies reveal a unique dosage requirement for telomere length maintenance by TERT; despite haploinsufficiency for the maintenance of long telomeres,
mTert
+/- mice retain minimal telomere DNA at all chromosome ends and do not exhibit the infertility typical of telomerase-deficient strains. Unlike the long (>50 kbp) average telomere lengths of wild-type laboratory mice,
mTert
+/- animals mice possess short telomere lengths similar to humans and wild-derived mice. Unexpectedly,
mTert
+/- mice are ersatz carriers for genetic instability, because their mating led to accelerated genetic instability and infertility in null progeny. Thus, limiting TERT levels play a key role in the maintenance of genome integrity, with important ramifications for the maintenance of short telomeres in human cancer and aging.
Blockade of Attachment and Fusion Receptors Inhibits HIV-1 Infection of Human Cervical Tissue
Authors: Hu Qinxue , Frank Ines , Williams Vennansha , Santos John J. , Watts Patricia , Griffin George E. , Moore John P. , Pope Melissa , Shattock Robin J.
Published: April 2004

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell–specific intercellular adhesion molecule–grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3+HLA-DR− and CD3−HLA-DR+ cells, with a significant proportion of the latter also expressing dendritic cell–specific intercellular adhesion molecule–grabbing integrin. Bead depletion studies demonstrated that such HLA-DR+ cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.
Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA
Authors: Nelson Celeste M. , Pirone Dana M. , Tan John L. , Chen Christopher S.
Published: April 2004

Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.
Network-based regulatory pathways analysis
Authors: Xiong Momiao , Zhao Jinying , Xiong Hao
Published: April 2004

Motivation: A useful approach to unraveling and understanding complex biological networks is to decompose networks into basic functional and structural units. Recent application of convex analysis to metabolic networks leads to the development of network-based metabolic pathway analysis and the decomposition of metabolic networks into metabolic extreme pathways that are true functional units of metabolic systems. Metabolic extreme pathways are derived from limited knowledge of the metabolic networks, but provide an integrated predictive description of metabolic networks. It is important to extend the concept of network-based metabolic pathways to genetic networks and develop mathematical procedures for network-based regulatory pathway analysis.

Results: We have established Kirchhoff's first law in genetic networks and introduced a concept of gene flows using matrix decomposition method. The Kirchhoff's first law provides the theoretical foundations for mathematical framework for development defining network-based regulatory pathways, and applying convex analysis in decomposing the genetic networks into regulatory extreme pathways. We presented a new approach to characterize the extreme pathway and developed a new algorithm for identifying a set of extreme pathways. Convex analysis and extreme pathway structure provide a unified framework for functional and structural analysis of metabolic and genetic networks, which will increase our ability to analyze, interpret and predict the function of metabolic and genetic networks. The proposed models for network-based regulatory pathway analysis have been applied to apoptosis regulatory network.
Probabilistic nucleotide assembling method for sequencing by hybridization
Authors: Endo Takaho A.
Published: April 2004

Motivation: Developing a new method of assembling small sequences based on sequencing by hybridization with many positive and negative faults. First, an interpretation of a generic traveling salesman problem is provided (i.e. finding the shortest route for visiting many cities), using genetic algorithms. Second, positive errors are excluded before assembly by a sanitization process.

Results: The present method outperforms those described in previous studies, in terms of both time and accuracy.

Availability:  http://kamit.med.u-tokai.ac.jp/~takaho/sbh/index.html
Sequence patterns derived from the automated prediction of functional residues in structurally-aligned homologous protein families
Authors: Miguel Ricardo Núñez
Published: April 2004

Motivation: Most proteins have evolved to perform specific functions that are dependent on the adoption of well-defined three-dimensional (3D) structures. Specific patterns of conserved residues in amino acid sequences of divergently evolved proteins are frequently observed; these may reflect evolutionary restraints arising both from the need to maintain tertiary structure and the requirement to conserve residues more directly involved in function. Databases of such sequence patterns are valuable in identifying distant homologues, in predicting function and in the study of evolution.

Results: A fully automated database of protein sequence patterns, Functional Protein Sequence Pattern Database (FPSPD), has been derived from the analysis of the conserved residues that are predicted to be functional in structurally aligned homologous families in the HOMSTRAD database. Environment-dependent substitution tables, evolutionary trace analysis, solvent accessibility calculations and 3D-structures were used to obtain the FPSPD. The method yielded 3584 patterns that are considered functional and 3049 patterns that are probably functional. FPSPD could be useful for assigning a protein to a homologous superfamily and thereby providing clues about function.

Availability: FPSPD is available at http://www-cryst.bioc.cam.ac.uk/~fpspd/
GEPIS—quantitative gene expression profiling in normal and cancer tissues
Authors: Zhang Yan , Eberhard David A. , Frantz Gretchen D. , Dowd Patrick , Wu Thomas D. , Zhou Yan , Watanabe Colin , Luoh Shiuh-Ming , Polakis Paul , Hillan Kenneth J. , Wood William I. , Zhang Zemin
Published: April 2004

Motivation: Expression profiling in diverse tissues is fundamental to understanding gene function as well as therapeutic target identification. The vast collection of expressed sequence tags (ESTs) and the associated tissue source information provides an attractive opportunity for studying gene expression.

Results: To facilitate EST-based expression analysis, we developed GEPIS (gene expression profiling in silico), a tool that integrates EST and tissue source information to compute gene expression patterns in a large panel of normal and tumor samples. We found EST-based expression patterns to be consistent with published papers as well as our own experimental results. We also built a GEPIS Regional Atlas that depicts expression characteristics of all genes in a selected genomic region. This program can be adapted for large-scale screening for genes with desirable expression patterns, as illustrated by our large-scale mining for tissue- and tumor-specific genes.

Availability: The email server version of the GEPIS application is freely available at http://share.gene.com/share/gepis. An interactive version of GEPIS will soon be freely available at http://www.cgl.ucsf.edu/Research/genentech/gepis/. The source code, modules, data and gene lists can be downloaded at http://share.gene.com/share/gepis

Supplementary information: Supplementary tables and figures are available at http://www.cgl.ucsf.edu/Research/genentech/gepis/
The NAD+-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication
Authors: Pappas Donald L. , Frisch Ryan , Weinreich Michael
Published: April 2004

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.
Distinct aerobic and hypoxic mechanisms of HIF-α regulation by CSN5
Authors: Bemis Lynne , Chan Denise A. , Finkielstein Carla V. , Qi Lin , Sutphin Patrick D. , Chen Xiaojiang , Stenmark Kurt , Giaccia Amato J. , Zundel Wayne
Published: April 2004

Mammalian oxygen homeostasis is dependent on the HIF family of transcription factors. The CSN subunit, CSN5, binds both the CODD of HIF-1α and the pVHL tumor suppressor. High CSN5 expression generates a pVHL-independent form of CSN5 that stabilizes HIF-1α aerobically by inhibiting HIF-1α prolyl-564 hydroxylation. Aerobic CSN5 association with HIF-1α occurs independently of the CSN holocomplex, leading to HIF-1α stabilization independent of Cullin 2 deneddylation. CSN5 weakly associates with HIF-1α under hypoxia, but is required for optimal hypoxia-mediated HIF-1α stabilization. These results indicate that CSN5 regulates aerobic as well as hypoxic HIF-1α stability by different mechanisms during oncogenesis.
Systematic Insertional Mutagenesis of a Streptomycete Genome: A Link Between Osmoadaptation and Antibiotic Production
Authors: Bishop Amy , Fielding Sue , Dyson Paul , Herron Paul
Published: April 2004

The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces up to fivefold greater antibiotic yields than the wild-type strain.
Comparison of Human Chromosome 21 Conserved Nongenic Sequences (CNGs) With the Mouse and Dog Genomes Shows That Their Selective Constraint Is Independent of Their Genic Environment
Authors: Dermitzakis Emmanouil T. , Kirkness Ewen , Schwarz Scott , Birney Ewan , Reymond Alexandre , Antonarakis Stylianos E.
Published: April 2004

The analysis of conservation between the human and mouse genomes resulted in the identification of a large number of conserved nongenic sequences (CNGs). The functional significance of this nongenic conservation remains unknown, however. The availability of the sequence of a third mammalian genome, the dog, allows for a large-scale analysis of evolutionary attributes of CNGs in mammals. We have aligned 1638 previously identified CNGs and 976 conserved exons (CODs) from human chromosome 21 (Hsa21) with their orthologous sequences in mouse and dog. Attributes of selective constraint, such as sequence conservation, clustering, and direction of substitutions were compared between CNGs and CODs, showing a clear distinction between the two classes. We subsequently performed a chromosome-wide analysis of CNGs by correlating selective constraint metrics with their position on the chromosome and relative to their distance from genes. We found that CNGs appear to be randomly arranged in intergenic regions, with no bias to be closer or farther from genes. Moreover, conservation and clustering of substitutions of CNGs appear to be completely independent of their distance from genes. These results suggest that the majority of CNGs are not typical of previously described regulatory elements in terms of their location. We propose models for a global role of CNGs in genome function and regulation, through long-distance cis or trans chromosomal interactions.
Shorter side chains optimize helix–helix packing
Authors: Jiang Sulin , Vakser Ilya A.
Published: April 2004

A systematic study of helix–helix packing in a comprehensive database of protein structures revealed that the side chains inside helix–helix interfaces on average are shorter than those in the noninterface parts of the helices. The study follows our earlier study of this effect in transmembrane helices. The results obtained on the entire database of protein structures are consistent with those obtained on the transmembrane helices. The difference in the length of interface and noninterface side chains is small but statistically significant. It indicates that helices, if viewed along their main axis, statistically are not circular, but have a flattened interface. This effect brings the helices closer to each other and creates a tighter structural packing. The results provide an interesting insight into the aspects of protein structure and folding.