The ability of sterol carrier protein-2 (SCP-2) to interact with long chain fatty acyl-CoAs was examined. SCP-2 bound fluorescent fatty acyl-CoAs at a single site with high affinity. K d values for cis-and trans-parinaroylCoA were 4.5 and 2.8 nM, respectively. Saturated 10 -18-carbon and unsaturated 14 -20-carbon fatty acyl-CoAs displaced SCP-2-bound fluorescent ligand. Oleoyl-CoA and oleic acid (but not coenzyme A) significantly altered SCP-2 Trp 50 emission and anisotropy decay, thereby increasing SCP-2 rotational correlation time, SCP-2 hydrodynamic radius, and SCP-2 Trp 50 remaining anisotropy up to 1.7-, 1.2-, and 1.3-fold, respectively. These changes were not accompanied by significant alterations in protein secondary structure as determined by circular dichroism. Finally, SCP-2 differentially altered the fluorescence emission and anisotropy decays of bound cis-and trans-parinaroyl-CoA. Both fluorescent fatty acyl-CoAs were located within a very ordered (limited cone angle of rotation) environment within SCP-2, as shown by a remaining anisotropy of 0.365 and 0.361 and a wobbling cone angle of 12 and 13°, respectively. These anisotropy values were very close to those of such ligands in a propylene glass. However, the rotational relaxation times exhibited by SCP-2-bound cis-and trans-parinaroyl-CoA, 8.4 -8.8 ns, were longer than those for the corresponding free fatty acid, 7.5-6.6 ns. These data show for the first time that SCP-2 is a fatty acylCoA-binding protein.Long chain fatty acyl-CoAs are not only important in the intermediary metabolism of fatty acids and the production of cellular energy; they are also potent regulators of many intracellular functions including neutrophil signaling, Ca 2ϩ signaling, insulin release, protein acylation, protein kinase activation, and gene transcription (reviewed in Ref. 1). The fatty acyl-CoAs are extremely potent, with regulatory activities occurring in the low nanomolar range (2-6).Cellular concentrations of fatty acyl-CoAs range from 2 to 248 nmol/g of protein, depending on tissue, and are as high as 110 -150 M in the cytosol of normal liver (reviewed in Ref. 1). Furthermore, fatty acyl-CoA levels can vary over a 20-fold range, depending on nutrition, drugs, endocrine status, or oxygenation (reviewed in Ref. 1). Cytosolic fatty acyl-CoAs are found bound to cell membranes (7) and to small nonenzymatic cytosolic proteins.It was recognized over 20 years ago that cytosolic fatty acidbinding proteins, representing 2-6% of cytosolic protein, can bind fatty acyl-CoAs (reviewed in Ref. 1). More than 17 members of this family have thus far been identified, with most of them having low micromolar affinities for fatty acyl-CoAs. More recently a totally different fatty acyl-CoA-binding protein (ACBP), 1 unrelated to the fatty acid-binding protein superfamily, was reported (8). ACBP has higher affinity for fatty acylCoAs than the fatty acid-binding proteins (9, 10).In contrast to the ACBP, the cellular sterol carrier protein-2 (SCP-2) has long been associated with the metabolism...

Although native rat liver fatty acid binding protein (L-FABP) is composed of isoforms differing in isoelectric point, their comparative structure and function are unknown. These properties of apo- and holo-L-FABP isoforms were resolved by circular dichroism, time-resolved fluorescence spectroscopy, and binding/displacement of fluorescent ligands. Both apo-isoforms had similar hydrodynamic radii of 18.5 A, but apo-isoform I had a greater alpha-helical content and exhibited a longer Tyr lifetime, indicative of secondary and tertiary structural differences from isoform II. Isoforms I and II both had two fatty acid or fatty acyl CoA binding sites. Ligand binding decreased the isoform hydrodynamic radii by 3-4 A and increased Tyr rotational motions in a more restricted range. Fatty acyl CoAs were more effective than fatty acids in altering the isoform structures. Scatchard analysis showed that both isoforms bound cis- parinaric acid with high affinity (Kd values 41 and 60 nM, respectively) and bound trans-parinaric acid with 2- and 7-fold, respectively, higher affinity than for cis-parinaric acid. In contrast, isoform I had higher affinity for cis- and trans-parinaroyl CoAs (Kd values of 33 and 14 nM) than did isoform II (Kd values of 110 and 97 nM), thereby resulting in biphasic plots of parinaroyl-CoA binding to native L-FABP. Finally, displacement studies indicated that each isoform displayed distinct specificities for fatty acid/fatty acyl CoA chain length and unsaturation. Thus, rat L-FABP isoforms differ markedly in both structure and ligand binding function.

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14-22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and L-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp50 and bound cis-parinaric acid was determined to be 40 A. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp50. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.

BackgroundA precise anatomical understanding of the thoracic paravertebral space (TPVS) is essential to understanding how an injection outside this space can result in paravertebral spread. Therefore, we aimed to clarify the three-dimensional (3D) structures of the TPVS and adjacent tissues using micro-CT, and investigate the potential routes for nerve blockade in this area.MethodsEleven embalmed cadavers were used in this study. Micro-CT images of the TPVS were acquired after phosphotungstic acid preparation at the mid-thoracic region. The TPVS was examined meticulously based on its 3D topography.ResultsMicro-CT images clearly showed the serial topography of the TPVS and its adjacent spaces. First, the TPVS was a very narrow space with the posterior intercostal vessels very close to the pleura. Second, the superior costotransverse ligament (SCTL) incompletely formed the posterior wall of the TPVS between the internal intercostal membrane and vertebral body. Third, the retro-SCTL space broadly communicated with the TPVS via slits, costotransverse space, intervertebral foramen, and erector spinae compartment. Fourth, the costotransverse space was intersegmentally connected to the adjacent retro-SCTL space.ConclusionsA non-destructive, multi-sectional approach using 3D micro-CT more comprehensively demonstrated the real topography of the intricate TPVS than previous cadaver studies. The posterior boundary and connectivity of the TPVS provides an anatomical rationale for the notion that paravertebral spread can be achieved with an injection outside this space.

The paravertebral spread that occurs after erector spinae plane block may be volume-dependent. This cadaveric study was undertaken to compare the extent of paravertebral spread with erector spinae plane block using different dye volumes. After randomization, twelve erector spinae plane blocks were performed bilaterally with either 10 ml or 30 ml of dye at the level of T5 in seven unembalmed cadavers except for two cases of unexpected pleural puncture using the 10 ml injection. Direct visualization of the paravertebral space by endoscopy was performed immediately after the injections. The back regions were also dissected, and dye spread and nerve involvement were investigated. A total of five 10 ml injections and seven 30 ml injections were completed for both endoscopic and anatomical evaluations. No paravertebral spread was observed by endoscopy after any of the 10-ml injections. Dye spread to spinal nerves at the intervertebral foramen was identified by endoscopy at adjacent levels of T5 (median: three levels) in all 30 ml injections. In contrast, the cases with two, four, and three out of five were stained at only the T4, T5, and T6 levels, respectively, with the 10 ml injection. Upon anatomical dissection, all blocks were consistently associated with posterior and lateral spread to back muscles and fascial layers, especially with the 30 ml injections, which showed greater dye expansion. In one 30 ml injection, sympathetic nerve involvement and epidural spread were observed at the level of the injection site. Although paravertebral spread following erector spinae plane block increased in a volume-dependent manner, this increase was variable and not pronounced. As the injectate volume increased for the erector spinae blocks, the injectate spread to the back muscles and fascial layers seemed to be predominantly increased compared with, the extent of paravertebral spread.