Low flow promotes instent intimal hyperplasia. Comparison with lumen loss in balloon-injured and uninjured vessels and the effects of the antioxidant pyrrolidine dithiocarbamate

Low flow (LF) promotes late lumen loss after angioplasty by exacerbating inward remodelling through redox-sensitive mechanisms. Stents eliminate inward remodelling and the effect of LF on in-stent restenosis is uncertain. We performed over-sized (1.3-1.5:1) stenting (S) and balloon injury (in the same vessel, B) to the carotid arteries of cholesterol-fed rabbits and compared 28-day late lumen loss with that in an uninjured segment in the same vessel (U). Vessels (n = 5 animals per group) were subjected to high (H), normal (N) and low (L) flow in animals fed either vehicle (V) or the antioxidant pyrrolidine dithiocarbamate, PDTC (P). LF significantly increased in-stent neointima formation relative to normal and high flow (SLV 0.72 +/- 0.07 mm(2) versus SNV 0.43 +/- 0.08 mm(2) versus SHV 0.28 +/- 0.04 mm(2), P < 0.05). However, LF resulted in greater lumen loss in segments from the same vessel subject to balloon injury (lumen SLV 5.18 +/- 0.40 mm(2) and SNV 5.32 +/- 0.40 mm(2) versus BLV 1.28 +/- 0.33 mm(2) and BNV 2.19 +/- 0.28 mm(2)), by greater enhancement of inward remodelling. In addition, inward remodelling and lumen loss due to LF were greater in balloon-injured segments than in adjacent uninjured segments where shear homeostatic remodelling occurs (lumen BLV 1.28 +/- 0.33 mm(2) versus ULV 1.52 +/- 0.22 mm(2)). Lastly, while PDTC effectively reduced intima formation and inward remodelling due to LF in balloon-injured vessels there was no effect on flow-dependent neointima formation in stented vessels. We conclude that LF accentuates in-stent neointima formation, but that flow-dependent lumen loss after stenting is less than that after balloon injury. When LF is present lumen loss can be minimised by antioxidants or stenting.     

Inhibition of Intra-Abdominal Adhesions: A Comparison of Hemaseel APR and Cryoprecipitate Fibrin Glue

Our previous studies demonstrated fibrin glue (FG) prepared from cryoprecipitate (cryo) inhibits intra-abdominal adhesions in rats. A new FG, Hemaseel APR, is Food and Drug Administration (FDA) approved for hemostasis during cardiac surgery and splenic trauma. This study was undertaken to determine if Hemaseel FG prevents intra-abdominal adhesions, and to compare it to cryo FG. Forty-five rats underwent laparotomy. Bilateral peritoneal-muscular defects were created. Polypropylene mesh was sewn into each defect with a running silk suture. The bowel was abraded with gauze. The rats were then randomized to mesh covered with Hemaseel FG, cryo FG, or control. On postoperative day 7, the severity of adhesions were graded by percentage of mesh covered by adhesion (0-100%) and degree of adhesion (0-3). The mean percentage of mesh covered by adhesion was 9% for Hemaseel FG, 43% for cryo FG (p = .005), and 65% for the controls (p &lt; .0001). The mean density adhesion score was 0.5 for Hemaseel FG, 1.2 for cryo FG (p = .04), and 2.1 for the controls (p &lt; .0001). In the Hemaseel FG group, 77% of patches had no adhesions, compared with 37% in the cryo FG group (p = .004) and 13% in the controls (p &lt; .0001). Thus, Hemaseel FG significantly decreases intra-abdominal adhesions, and is more effective than cryo FG.     

Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood

BACKGROUND: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability. METHODS: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables. RESULTS: Storage of blood samples for >1 week at 4 degrees C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex. CONCLUSION: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.     

Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue.

Nitrogenase in Rhodospirillum rubrum is inactivated in vivo by the covalent modification of the Fe protein with a nucleotide. The preparation of two modified peptides derived from proteolytic digestion of the inactive Fe protein is described. The modifying group is shown to be adenosine diphosphoribose, linked through the terminal ribose to a guanidino nitrogen of arginine. The structural features were established by using proton and phosphorus NMR, positive- and negative-ion fast atom bombardment mass spectrometry, and fast atom bombardment/collisionally activated decomposition mass spectrometry. Spectral methods along with chromatographic analysis and sequential degradation established the sequence of the modification site of Fe protein as Gly-Arg(ADR-ribose)-Gly-Val-Ile-Thr. This corresponds to the sequence in the Fe protein from Azotobacter vinelandii for amino acid residues 99 to 104.     

Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.

An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.     

[11C]5-HTP and microPET are not suitable for pharmacodynamic studies in the rodent brain

The PET tracer [¹¹C]5-hydroxytryptophan ([¹¹C]5-HTP), which is converted to [¹¹C]5-hydroxytryptamine ([¹¹C]5-HT) by aromatic amino acid decarboxylase (AADC), is thought to measure 5-HT synthesis rates. But can we measure these synthesis rates by kinetic modeling of [¹¹C]5-HTP in rat? Male rats were scanned with [¹¹C]5-HTP (60 minutes) after different treatments. Scans included arterial blood sampling and metabolite analysis. 5-HT synthesis rates were calculated by a two-tissue compartment model (2TCM) with irreversible tracer trapping or Patlak analysis. Carbidopa (inhibitor peripheral AADC) dose-dependently increased [¹¹C]5-HTP brain uptake, but did not influence 2TCM parameters. Therefore, 10 mg/kg carbidopa was applied in all subsequent study groups. These groups included treatment with NSD 1015 (general AADC inhibitor) or p-chlorophenylalanine (PCPA, inhibitor of tryptophan hydroxylase, TPH). In addition, the effect of a low-tryptophan (Trp) diet was investigated. NSD 1015 or Trp depletion did not affect any model parameters, but PCPA reduced [¹¹C]5-HTP uptake, and the k₃. This was unexpected as NSD 1015 directly inhibits the enzyme converting [¹¹C]5-HTP to [¹¹C]5-HT, suggesting that trapping of radioactivity does not distinguish between parent tracer and its metabolites. As different results have been acquired in monkeys and humans, [¹¹C]5-HTP-PET may be suitable for measuring 5-HT synthesis in primates, but not in rodents.