Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.     

Use of a novel triple-tracer approach to assess postprandial glucose metabolism

Numerous studies have used the dual-tracer method to assess postprandial glucose metabolism. The present experiments were undertaken to determine whether the marked tracer nonsteady state that occurs with the dual-tracer approach after food ingestion introduces error when it is used to simultaneously measure both meal glucose appearance (R(a meal)) and endogenous glucose production (EGP). To do so, a novel triple-tracer approach was designed: 12 subjects ingested a mixed meal containing [1-(13)C]glucose while [6-(3)H]glucose and [6,6-(2)H(2)]glucose were infused intravenously in patterns that minimized the change in the plasma ratios of [6-(3)H]glucose to [1-(13)C]glucose and of [6,6-(2)H(2)]glucose to endogenous glucose, respectively. R(a meal) and EGP measured with this approach were essentially model independent, since non-steady-state error was minimized by the protocol. Initial splanchnic glucose extraction (ISE) was 12.9% +/- 3.4%, and suppression of EGP (EGPS) was 40.3% +/- 4.1%. In contrast, when calculated with the dual-tracer one-compartment model, ISE was higher (P < 0.05) and EGPS was lower (P < 0.005) than observed with the triple-tracer approach. These errors could only be prevented by using time-varying volumes different for R(a meal) and EGP. Analysis of the dual-tracer data with a two-compartment model reduced but did not totally avoid the problems associated with marked postprandial changes in the tracer-to-tracee ratios. We conclude that results from previous studies that have used the dual-tracer one-compartment model to measure postprandial carbohydrate metabolism need to be reevaluated and that the triple-tracer technique may provide a useful approach for doing so.     

Need for gerodontology education in India

Objectives: To evaluate the status and need for education in Geriatric Dentistry in India. Materials and methods: The status of dental education in India was studied. India has 185 dental schools producing 12 000 dental graduates every year. At postgraduate level, there are nine recognised specialities in Dentistry, and approximately 58 institutions offer 1160 places for postgraduate degree courses. Dental education is shaped and administered by the Dental Council of India. Results: So far, Geriatric dentistry has not developed as a separate, independent speciality at postgraduate level. The undergraduate curriculum does not have any significant component of geriatric dentistry. The lack of training results in poor understanding of special needs of older adults in young graduates. Without adequate training and personal experience of growing old, young graduates may not be able to understand the physical, socio‐economic and psychological problems of the elderly and the complexities involved in treatment planning for patients with multiple chronic diseases and medication. Conclusions: It is emphasized that geriatric dentistry should be included in each of the pre‐clinical, para‐clinical and clinical subjects at the undergraduate level. Graduate students should be encouraged to treat elderly patients in clinics under supervision using a multi‐disciplinary approach. Also, postgraduate diploma and degree courses in geriatric dentistry should be developed to address the needs of the vast elderly population in India.     

CRSBP-1/LYVE-l-null mice exhibit identifiable morphological and functional alterations of lymphatic capillary vessels

CRSBP-1, a membrane glycoprotein, can mediate cell-surface retention of secreted growth factors containing CRS motifs such as PDGF-BB. CRSBP-1 has recently been found to be identical to LYVE-1, a specific marker for lymphatic capillary endothelial cells. The in vivo role of CRSBP-1/LYVE-1 is unknown. CRSBP-1-null mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic capillary vessels in certain tissues, marked by the constitutively increased interstitial-lymphatic flow and lack of typical irregularly-shaped lumens. The CRSBP-1 ligands PDGF-BB and HA enhance interstitial-lymphatic flow in wild-type mice but not in CRSBP-1-null animals.     

The MOSIX Direct File System Access Method for Supporting Scalable Cluster File Systems

MOSIX is a cluster management system that supports preemptive process migration. This paper presents the MOSIX Direct File System Access (DFSA), a provision that can improve the performance of cluster file systems by allowing a migrated process to directly access files in its current location. This capability, when combined with an appropriate file system, could substantially increase the I/O performance and reduce the network congestion by migrating an I/O intensive process to a file server rather than the traditional way of bringing the file's data to the process. DFSA is suitable for clusters that manage a pool of shared disks among multiple machines. With DFSA, it is possible to migrate parallel processes from a client node to file servers for parallel access to different files. Any consistent file system can be adjusted to work with DFSA. To test its performance, we developed the MOSIX File-System (MFS) which allows consistent parallel operations on different files. The paper describes DFSA and presents the performance of MFS with and without DFSA.     

Computer simulations improve university instructional laboratories

Laboratory classes are commonplace and essential in biology departments but can sometimes be cumbersome, unreliable, and a drain on time and resources. As university intakes increase, pressure on budgets and staff time can often lead to reduction in practical class provision. Frequently, the ability to use laboratory equipment, mix solutions, and manipulate test animals are essential learning outcomes, and "wet" laboratory classes are thus appropriate. In others, however, interpretation and manipulation of the data are the primary learning outcomes, and here, computer-based simulations can provide a cheaper, easier, and less time- and labor-intensive alternative. We report the evaluation of two computer-based simulations of practical exercises: the first in chromosome analysis, the second in bioinformatics. Simulations can provide significant time savings to students (by a factor of four in our first case study) without affecting learning, as measured by performance in assessment. Moreover, under certain circumstances, performance can be improved by the use of simulations (by 7% in our second case study). We concluded that the introduction of these simulations can significantly enhance student learning where consideration of the learning outcomes indicates that it might be appropriate. In addition, they can offer significant benefits to teaching staff.     

Phylo-VISTA: interactive visualization of multiple DNA sequence alignments

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu     

Electron microscope tomography of Balbiani Ring hnRNP substructure

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultra-structural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.Abbreviations BR Balbiani Ring - EMT electron microscope tomography - ESI electron spectroscopic imaging - hnRNP heterogeneous nuclear ribonucleoprotein - OA-B osmium ammine-B - kb kilobases by P.B. Moens     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.