Contusion spinal cord injury (SCI) animal models are used to study loss of muscle function and mass. However, parallels to the human condition typically have been confounded by spontaneous recovery observed within the first few post-injury weeks, partly because of free cage activity. We implemented a new rat model combining SCI with cast immobilization (IMM) to more closely reproduce the unloading conditions experienced by SCI patients. Magnetic resonance imaging was used to monitor hindlimb muscles' cross-sectional area (CSA) after SCI, IMM alone, SCI combined with IMM (SCI + IMM), and in controls (CTR) over a period of 21 days. Soleus muscle tetanic force was measured in situ on day 21, and hindlimb muscles were harvested for histology. IMM alone produced a decrease in triceps surae CSA to 63.9 -4.9% of baseline values within 14 days. In SCI, CSA decreased to 75.0 -10.5% after 7 days, and recovered to 77.9 -10.7% by day 21. SCI + IMM showed the greatest amount of atrophy (56.9 -9.9% on day 21). In all groups, muscle mass and soleus tetanic force decreased in parallel, such that specific force was maintained. Extensor digitorum longus (EDL) and soleus fiber size decreased in all groups, particularly in SCI + IMM. We observed a significant degree of asymmetry in muscle CSA in SCI but not IMM. This effect increased between day 7 and 21 in SCI, but also in SCI + IMM, suggesting a minor dependence on muscle activity. SCI + IMM offers a clinically relevant model of SCI to investigate the mechanistic basis for skeletal muscle adaptations after SCI and develop therapeutic approaches.
Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.
Bioassay studies have indicated that angiotensin I (Ang I), but not angiotensin II (Ang II), is degraded by the intact lung. The present study was an attempt to isolate and quantify pulmonary metabolites of Ang I. The pulmonary vascular bed of the rat was isolated and perfused at 4-6 ml/min (< 20 mm Hg) with oxygenated Krebs buffer containing 5% bovine serum albumin. [3H-Leu10]Ang I (3-10 ng) was administered, and pulmonary effluent samples were collected every 15 sec for 5 min. Peptides were isolated by Dowex chromatography and separated by thin layer chromatography. 3H-labeled peptides were eluted from the thin layer chromatography plate, and the Ang I and [des-Asp1]Ang I levels were estimated by RIA. These peptides were shown to be hydrolyzed by purified converting enzyme with the release of His-3H-Leu. About 25% of the [3H]Ang I administered was isolated as [3H-des-Asp1]Ang I, and this percentage increased to 33% after treatment with teprotide. These studies clearly demonstrate that [des-Asp1]Ang I is a major pulmonary metabolite of Ang I and suggest the presence of a pulmonary aminopeptidase which hydrolyzes Ang I but not Ang II.
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