Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus.

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.

Design and synthesis of Cyclopenta[g]quinazoline-based antifolates as inhibitors of thymidylate synthase and potential antitumor agents(,).

Following the development of raltitrexed, the synthesis of nonpolyglutamatable inhibitors of TS that do not use the reduced folate carrier (RFC) for cellular entry should provide compounds which overcome mechanisms of resistance to folate-based inhibitors of TS that are associated with decreased/altered folylpolyglutamate synthetase (FPGS) expression and/or an impaired RFC. Examination of a computer graphics model of the humanized Escherichia coli TS enzyme with quinazoline inhibitors of TS, such as 1 bound in the active site of the enzyme, suggested that conformational restriction introduced by bridging the C9 with C7 to form a pentacycle may be beneficial for binding to TS. That led to the synthesis of a series of potent cyclopenta[g]quinazoline-based inhibitors of the enzyme in which the glutamyl residue associated with classical antifolates was replaced with a variety of glutamate-derived ligands; the most potent inhibitor being the L-Glu-gamma-D-GluT(alpha) derivative 7j. In the mouse L1210:1565 cell line (mutant RFC), the majority of these compounds had activity equal or only slightly greater compared with the parental L1210 cell line, indicating a reduced dependence on the RFC for cellular uptake in the L1210 cell line.

Digestion and absorption of tube-feeding emulsions with different droplet sizes and compositions in the rat.

Assimilation of lipid nutrients depends on the efficiency of emulsified fat hydrolysis by digestive lipases. As shown in vitro, the activity of preduodenal and pancreatic lipases is governed by the physicochemical properties of emulsions. Thus the aim of this study was to evaluate in the rat how emulsions are digested and assimilated depending on their droplet size or solute composition. Fasted rats were intragastrically tube fed emulsions with different median droplet sizes (0.6 microns, fine; 22 microns, coarse) or solute composition (0.8 microns, complex fine) containing 14C-triolein and 3H-cholesterol. Two and 5 hours after feeding, fat-droplet size was measured in gastric and duodenal contents, and lipids were radioactively quantified in different compartments. In the stomach, the droplet size of the fine emulsions significantly increased to values (13 microns to 24 microns) comparable with those of the coarse emulsion (35 microns to 36 microns). In the duodenum, the droplet sizes of the three emulsions were in the range of 14 microns to 33 microns. After 2 hours, gastric triglyceride hydrolysis was significantly higher with the fine than with the coarse emulsion and was lower with the complex fine emulsion. Gastric emptying of fat was significantly different, with the following decreasing order: coarse, fine, and complex fine emulsion. In the small intestine, the fine and coarse emulsions were processed comparably, whereas the assimilation of the fine complex emulsion was significantly delayed. Calculations indicate that ingested fatty acids were distributed in the peripheral tissues at different rates with the same decreasing order. The fate of a lipophilic nutrient, cholesterol, was also markedly altered by the type of emulsion. These data support the concept that tube-fed emulsions with different droplet sizes and solute composition are digested differently and thus are metabolized differently.

Sepsis modifies the contribution of different organs to whole-body protein synthesis in rats.

1. Protein synthesis rate was measured in the liver, muscle, intestine and whole body of septic rats and pair-fed controls by administration of a large dose of L-[U14C]valine. Sepsis was induced by intravenous injection of live bacteria, and protein synthesis measurements were carried out 48 h later. 2. Septic rats exhibited a reduction in food intake to between 10 and 50% of the normal level on the 2 days after infection. Animals lost body weight and the relative organ weight was increased in liver, unchanged in intestine and decreased in skeletal muscle. 3. The fractional protein synthesis rate of the whole body excluding liver was increased by 19% in septic rats in comparison with pair-fed controls, but the absolute protein synthesis rate was similar in the two groups because of the low protein content of the infected group. 4. The fractional protein synthesis was increased in whole intestine and liver but was decreased in skeletal muscle. In muscle and liver, the difference between infected and pair-fed animals was more pronounced for the absolute than for the fractional protein synthesis rate. In intestine, the fractional protein synthesis rate was similarly increased in whole intestine and the muscular layer of ileum. This suggests different regulation of protein synthesis in skeletal and smooth muscles. 5. The present investigation shows a complete redistribution of protein synthesis in sepsis. Liver represents about a third of the whole-body protein synthesis instead of 15% and becomes predominant over synthesis of muscle.

Understanding the context for long-range planning in hospitals.

Using their own experience in an urban hospital of national reputation, the authors describe and illustrate some of the background issues for long-range planning decisions pointing out that managers must steer a clear course among a number of internal and external pressures. The expectations of trustees, philanthropists, reimbursing agencies, the university, medical staff, patients and the surrounding community may sometimes be at odds. Managers can use long-range planning as a tool to ease the hospital into changing modes of operation and to achieve, over a time, a smoother interaction among their several constituencies.