Synthesis and stability studies of 2',3',5'-tri-O-acetyl-2-amino(-N6-cyclopentyl)-1-deazaadenosines

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.     

Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation.

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.     

Prolactin stimulation of protein kinase C activity in rat aortic smooth muscle

Prolactin (PRL) activated protein kinase C (PKC) in a dose dependent manner in rat aortic smooth muscle. Aortic strips incubated with sub-nanomolar concentrations of ovine PRL for 25 min. at 37 degrees C showed a significant stimulation of PKC activity in both cytosolic and particulate fractions. This activation could be blocked using either anti-PRL antibodies or 1-(5- isoquinolinesulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The results further support the role of PKC in the signal transduction pathway for PRL action and suggest that this activation may be involved in vascular smooth muscle function.     

An insight into some relevant aspects concerning eriophyoid mites inhabiting forests, ornamental trees and shrubs

Worldwide a great variety of eriophyoid mites inhabit forest canopy trees and ornamental plants that are used in city parks, squares and boulevards. An analysis of the relevant bibliography portrays only a fragmentary knowledge and the majority of our information concerns the temperate zone. Three case studies are presented as examples of different approaches to solve problems connected with eriophyoid mites of forest and ornamental trees. The first example deals with eriophyoids of a temperate zone forest in a natural environment, focusing on conifers which represent the largest component. The second case study documents a possible approach to obtain greater knowledge and control of the bud mite species, Trisetacus juniperinus (Nalepa) on Cupressaceae. This is a harmless species in the natural environment which becomes a serious pest in nurseries and young stands of Cupressus sempervirens in the Mediterranean region. The final case study reports on long-term studies carried out in Poland on injurious eriophyoid species that are found in nurseries, city greenery and parks. This paper also discusses future perspectives for research on eriophyoid mites living on forest and ornamental plants.     

Computational modeling of the immune response to tumor antigens

Vaccination protocols designed to elicit anti-cancer immune responses have, many times, failed in producing tumor eradication and in prolonging patient survival. Usually in cancer vaccination, epitopes from one organism are included in the genome or linked with some protein of another in the hope that the immunogenic properties of the latter will boost an immune response to the former. However, recent results have demonstrated that injections of two different vectors encoding the same recombinant antigen generate high levels of specific immunity. Systematic comparison of the efficacy of different vaccination protocols has been hampered by technical limitations, and clear evidence that the use of multiple vectors has advantages over single carrier injections is lacking. We used a computational model to investigate the dynamics of the immune response to different anti-cancer vaccines based on randomly generated antigen/carrier compounds. The computer model was adapted for simulations to this new area in immunology research and carefully validated to the purpose. As a matter of fact, it reproduces a relevant number of experimental observations. The model shows that when priming and boosting with the same construct, competition rather than cooperation develops amongst T cell clones of different specificities. Moreover, from the simulations, it appears that the sequential use of multiple carriers may generate more robust anti-tumor immune responses and may lead to effective tumor eradication in a higher percentage of cases. Our results provide a rational background for the design of novel strategies for the achievement of immune control of cancer.     

Tomato transgenic lines and Tetranychus urticae: changes in plant suitability and susceptibility

A critical aspect dealing with the use of transgenic plants is the global evaluation of their environmental impact. The polyphagous mite Tetranychus urticae can be considered a suitable species to investigate unpredictable and undesirable effects on phytophagous arthropods. Three tomato near isogenic lines, that is, the cv. Riogrande (RIG), the transgenic lines RC332 (containing the Gox gene and showing high glucose oxidase activity), and MS498 (containing the KTI3 gene and exhibiting a high trypsin inhibition) were used in laboratory and greenhouse trials. Trichomes and contents of C and N of the leaves, differences in development and oviposition of T. urticae and damage caused were evaluated for each line. The laboratory trials evidenced that (1) the intrinsic rate of increase of two strains of T. urticae (T from tomato, B from bindweed), reared on the lower surface of tomato leaflets, was significantly lower in RIG than in transgenic lines and doubling time ranged between 6.9 and 11.6 days in the first and between 3.9 and 5.3 days in the latter; (2) the glandular four-lobed trichomes were always higher in RIG than in other genotypes; (3) the N leaf content was from 1.3 to 1.9 fold lower and the C/N ratio from 1.3 to 1.9 fold higher in RIG than in other lines. The greenhouse experiment, that lasted over a month and was performed by inducing an initially equal infestation of strain T, evidenced: (1) no significant difference between plant lines in the final mite infestation (motile stages per plant), nevertheless an almost double number of spider mites was counted in RC332; (2) a significantly higher percentage of damaged leaves and a significant higher average damage index on RC332 than on RIG (79% and 2.3 in the former, and 62% and 2.1 in the latter, respectively), even if in both transgenics a higher level of the most severe damages and a shorter time to approach them were observed; (4) a comparable number of mites causing the same damage level in all genotypes and a strong linear relation between the first four levels of damage and mite infestation. Although in the laboratory studies both transgenic lines enhanced the T. urticae population increase, the glasshouse studies were not as conclusive and they only suggest the possibility of any real difference between the transgenic and non-transgenic genotypes.     

Computer-aided design of biological circuits using TinkerCell

Synthetic biology is an engineering discipline that builds on modeling practices from systems biology and wet-lab techniques from genetic engineering. As synthetic biology advances, efficient procedures will be developed that will allow a synthetic biologist to design, analyze, and build biological networks. In this idealized pipeline, computer-aided design (CAD) is a necessary component. The role of a CAD application would be to allow efficient transition from a general design to a final product. TinkerCell is a design tool for serving this purpose in synthetic biology. In TinkerCell, users build biological networks using biological parts and modules. The network can be analyzed using one of several functions provided by TinkerCell or custom programs from third-party sources. Since best practices for modeling and constructing synthetic biology networks have not yet been established, TinkerCell is designed as a flexible and extensible application that can adjust itself to changes in the field.     

Chemical Composition and Antimicrobial Activity of the Essential Oils from Several Hypericum Taxa (Guttiferae) Growing in Central Italy (Appennino Umbro‐Marchigiano)

The chemical composition of the essential oils of nine taxa from seven sections of Hypericum L. (Guttiferae; H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. montanum, H. richeri subsp. richeri, H. hyssopifolium, H. hirsutum, H. hircinum subsp. majus, and H. tetrapterum) occurring in central Italy (Appennino Umbro‐Marchigiano) was analyzed by GC/FID and GC/MS. A total of 186 compounds were identified in the different species and subspecies, accounting for 86.9–92.8% of the total oils. The major fraction of the oil was always represented by sesquiterpene hydrocarbons (30.3–77.2%), while quantitative differences occurred between the other classes of volatiles depending on the taxa considered. Chemical composition of the nine Hypericum entities with respect to the taxonomical classification was discussed. Essential oils obtained from six taxa, i.e., H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. richeri subsp. richeri, H. hirsutum and H. tetrapterum, were also tested for their antimicrobial properties against five different microbial strains by the broth‐microdilution method, and they were found to have significant activity (expressed as MIC) on B. subtilis, moderate activity on C. albicans and S. aureus, and weak activity on E. coli and E. faecalis, the most active being those from H. hirsutum, H. richeri subsp. richeri, and H. tetrapterum.     

Conservation analysis of large biochemical networks

MOTIVATION: Large biochemical networks pose a unique challenge from the point of view of evaluating conservation laws. The computational problem in most cases exceeds the capability of available software tools, often resulting in inaccurate computation of the number and form of conserved cycles. Such errors have profound effects on subsequent calculations, particularly in the evaluation of the Jacobian which is a critical quantity in many other calculations. The goal of this paper is to outline a new algorithm that is computationally efficient and robust at extracting the correct conservation laws for very large biochemical networks. RESULTS: We show that our algorithm can perform the conservation analysis of large biochemical networks, and can evaluate the correct conserved cycles when compared with other similar software tools. Biochemical simulators such as Jarnac and COPASI are successful at extracting only a subset of the conservation laws that our algorithm can. This is illustrated with examples for some large networks which show the advantages of our method.