An optimized protocol for first strand cDNA synthesis from laser capture microdissected tissue.
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DNA, Complementary↗
Biomedical subjects
S Boylan
Publications and source records attributed to S Boylan.
Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation.
Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Delta1-91RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the RII PKA isoforms.
Catalytic Domain↗
Fractures in cryopreserved elastic arteries.
The aim of this study was to define the conditions under which macroscopic fractures occur in vascular tissue during cryopreservation and to develop a practical cryopreservation method that prevents fracturing. The common carotid artery of the rabbit was subjected to a cryopreservation process that has been optimised for retention of in vitro function and cytological structure. This involves the stepwise addition and subsequent removal of dimethyl sulfoxide using a calculated protocol that avoids osmotic injury and minimises toxic action, controlled cooling, storage at -180 degrees C, and rapid warming. Seventy-five percent of such arteries were grossly fractured. The cooling and warming conditions were systematically varied to determine when in the cooling/storage/warming process the fractures occurred. Differential scanning calorimetry was then used to identify any corresponding thermal events. It was found that the fractures occurred as the temperature range -150 to -100 degrees C was traversed during the warming phase of the process. The glass transition temperature of a maximally freeze-concentrated solution of the cryoprotectant used was found to be -123 degrees C. Reducing the warming rate between the storage temperature (-180 degrees C) and -100 degrees C to < 50 degrees C/min prevented the fractures. Subsequent thawing could then be carried out rapidly in a 37 degrees C water bath without risk of fracture. We suggest that the fractures probably result from the thermal stresses created by rapid warming of the vitreous material that is produced by freeze-concentration of the aqueous phase. Relatively slow warming to -100 degrees C, at which temperature the vitreous material has softened, reduces these stresses and avoids the fractures.
Animals↗
Cryopreservation of rabbit corneas in dimethyl sulfoxide.
PURPOSE: To minimize the injury to endothelial cells during cryopreservation of rabbit corneas with dimethyl sulfoxide. METHODS: Rabbit corneas were cryopreserved using 20% wt/wt dimethyl sulfoxide (Me2SO), added and removed in stages to maintain the osmotically induced excursions in cell volume to within +/-40% of their isotonic volume. The vehicle solution, cooling rate, and conditions of storage used were those already reported to be optimal for endothelial cell survival after exposure to low temperatures. Survival was assessed by confocal microscopy with vital staining and by the ability of the endothelium to control stromal hydration during 3 hours of normothermic perfusion. The effect of temperature of addition and removal of Me2SO (room temperature [RT] or 2 degrees C) on endothelial viability also was measured. RESULTS: After thawing, all the cryopreserved corneas appeared structurally intact when assessed by vital staining and could limit stromal swelling during subsequent normothermic perfusion. Analysis of the rate of stromal swelling during the first 1.5 hours of normothermic perfusion indicated a substantial benefit when the Me2SO was removed at RT. Adding and removing the Me2SO at RT, which allowed a briefer exposure to Me2SO before cooling, resulted in better structural integrity of the endothelial layer than when the addition of cryoprotectant took place on ice. CONCLUSIONS: These results demonstrate the importance of osmotic stresses in the generation of injury to corneal endothelium during cryopreservation and the possibility of eventual successful cryopreservation of this tissue.
Animals↗
The effect of cooling rate and temperature on the toxicity of ethylene glycol in the rabbit internal carotid artery.
The smooth muscle and vascular endothelium of small elastic arteries (the rabbit common carotid artery) are injured by exposure to 40% ethylene glycol (EG) at 4 degrees C, and additional damage occurs when the arteries are cooled without freezing to -20 degrees C. This paper reports attempts to reduce this injury by altering the cooling rate and temperature of exposure to the cryoprotectant. Very slow cooling (0.1 degree C/min) removed all residual smooth muscle and endothelial function when assessed in vitro after rewarming and removal of the cryoprotectant. Very rapid cooling to -20 degrees C also increased the injury, both to the endothelium and to the smooth muscle. Reducing the temperature of exposure to 40% EG from +4 degrees C to -20 degrees C had no beneficial effect on the smooth muscle but enabled the vascular endothelium to retain some functional activity. These data suggest that the mechanism responsible may be related to the physical properties of ethylene glycol rather than to a biochemical interaction with metabolic processes, and that it is a mechanism which is highly specific for the cell types involved. It also underlines the difficulties involved in the successful cryopreservation of complex tissues and organs.
Animals↗
Ethylene glycol permeation and toxicity in the rabbit common carotid artery.
The rate of permeation of ethylene glycol (EG) and the maximum concentration that can be tolerated without functional damage was measured in the rabbit common carotid artery. Pairs of arteries were perfused on ice, one (the control) with a high K+ balanced salt solution containing 100 mM TES (CPTES), and the other with ethylene glycol/CPTES solutions. The concentration of EG was increased in a stepwise manner in order to reduce osmotically induced changes in endothelial cell volume. The final concentration was 10, 20, or 40% EG (w/w). After exposure for 20 min, the EG was then removed at room temperature using stepwise decreasing concentrations of ethylene glycol in the presence of 3% mannitol. After this, the contractile function of the smooth muscle was tested at 37 degrees C with noradrenaline and the integrity of the endothelium was assessed structurally by vital staining and functionally by its capacity to produce endothelium-derived relaxation factor in response to administration of acetylcholine. The tissue concentration reached 8.6% after 30 min of exposure to 10% EG. The contractile function of the smooth muscle was unaffected by EG at all concentrations. There was a significant (50%) reduction in the ACh-induced relaxation of contracted arteries after exposure to 40% EG (P < 0.02) but this was not associated with any detectable loss of cells or damage to the endothelium. It was concluded that EG warrants further investigation as a cryoprotectant for blood vessels.
Animals↗
Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties.
A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.
1,4-alpha-Glucan Branching Enzyme↗