Impact of prestorage leucoreduction of autologous whole blood on length of hospital stay with a subgroup analysis in bilateral hip arthroplasty.

BACKGROUND: Although prestorage leucoreduction (LR) of blood components for transfusion has gained favour around the world, evidence of its beneficial clinical effects is ambiguous. STUDY DESIGN AND METHODS: To reveal whether leucocytes and/or platelets in transfused blood are related to transfusion-related adverse effects, a prospective randomized crossover study was performed on patients who donated autologous blood prior to elective surgery. Among 1487 primary enrolees, a total of 192 patients undergoing two-stage, bilateral total hip arthroplasty were randomized to receive autologous blood that was either prestorage leucoreduced, or not, for the first procedure. For the second procedure, each patient was crossed over to receive alternatively processed autologous blood. Length of hospital stay served as a primary end-point, with perioperative infectious/thrombotic complications, pre- and postoperative laboratory values, and body temperature serving as secondary endpoints. RESULTS: No significant differences emerged between prestorage LR and non-LR cohorts in length of hospital stay, as well as perioperative infectious/thrombotic complications, postoperative body temperature and duration of fever. Postoperative laboratory values including white blood cell counts and C-reactive protein levels had no significant differences. CONCLUSION: This study could not prove any superiority of prestorage LR over non-LR for autologous whole blood among patients who underwent total hip arthroplasty.

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN.

The tumor suppressor PTEN is a phosphatase with sequence similarity to the cytoskeletal protein tensin. Here the cellular roles of PTEN were investigated. Overexpression of PTEN inhibited cell migration, whereas antisense PTEN enhanced migration. Integrin-mediated cell spreading and the formation of focal adhesions were down-regulated by wild-type PTEN but not by PTEN with an inactive phosphatase domain. PTEN interacted with the focal adhesion kinase FAK and reduced its tyrosine phosphorylation. Overexpression of FAK partially antagonized the effects of PTEN. Thus, PTEN phosphatase may function as a tumor suppressor by negatively regulating cell interactions with the extracellular matrix.

Global variation in G+C content along vertebrate genome DNA. Possible correlation with chromosome band structures.

The global, rather than local, variation in G+C content along the nuclear DNA sequences of various organisms was studied using GenBank sequence data. When long DNA sequences of the genomes of Escherichia coli and Saccharomyces cerevisiae were examined, the levels of their G+C content (G+C%) were found to be within a narrow range around that of the whole genome. The G+C% levels for sequences of vertebrate genomes, however, were found to cover a wide range, showing that their genome is a mosaic of sequences with different G+C% levels, in each of which the sequence is fairly homogeneous in its G+C% for a very long distance. Through surveying a human genetic map and GenBank DNA sequences, the global variations in G+C% along the human genome DNA were found to be correlated with chromosome band structures.

Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module.

The ninth type III module of murine fibronectin was expressed in E. coli and folded into a compact homogeneous monomer whose unfolding and refolding were then investigated by fluorescence, circular dichroism, calorimetry and electron microscopy. The isolated module is unusually labile under physiological conditions. When heated at 1 deg. C/minute it exhibits an irreversible endothermic transition between 35 and 42 degrees C depending on the protein concentration. The transition is accompanied by changes in secondary and tertiary structure with partial exposure of the single tryptophan and increased binding of the hydrophobic probe, 1,8-anilinonaphthalene-sulfonate. The partially unfolded intermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain substantial amounts of beta sheet structure. The multimers melt and dissociate reversibly in a second endothermic transition between 60 and 90 degrees C also depending on the protein concentration. This second transition destroys the remaining secondary structure and further exposes the tryptophan. Visualization of negatively stained specimens in the electron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of approximately 10 nm width and indeterminate length. A subdomain swapping mechanism is proposed in which beta strands from one partially unfolded molecule interact with complementary regions of another to form oligomers and polymers. The possibility that similar interactions could play a role in the formation of fibrils by fibronectin in vivo is discussed.

Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure.

We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.

The ActR-I activin receptor protein is expressed in notochord, lens placode and pituitary primordium cells in the mouse embryo.

ActR-I is a type I serine/threonine kinase receptor which has been shown to bind activin and bone morphogenetic proteins (BMPs). To study the function of ActR-I, we have generated novel monoclonal antibodies that specifically recognize the extracellular domain of mouse ActR-I. We examined the level of ActR-I protein during mouse development by immunohistochemistry. We found that in the embryonic body, ActR-I protein first appears in a restricted part of the primitive streak region and is present throughout the length of notochord. Furthermore, ActR-I protein is expressed in the facial sensory organ primordia, including eye area, otic vesicle and olfactory placode, which all contain invaginating ectoderm. In addition, ActR-I is produced in pituitary primordium (Rathke's pouch), mammary buds and the epithelial layer of branchial arches. Interestingly, in the lens placodes and in early Rathke's pouch, ActR-I protein is transiently localized at the apical surface of the epithelial cells, indicating the presence of an apical-basal asymmetry in these cells.

Nucleotide sequence and molecular evolution of mouse retrovirus-like IAP elements.

We determined the nucleotide (nt) sequences of cDNA and genomic clones for murine intracisternal type A particle (IAP) elements, which are retrovirus-like repetitive sequences in rodent genomes. The nucleotide sequence of the cDNA resembled that of retrovirus RNA genomes in its lack of the U5 sequence within the 3' long terminal repeat. By sequence comparison of our clones with reported rodent IAP elements, we located the probable gag, pol and env gene regions. The sequences for the pol, env and the 3' two-thirds of the gag region were conserved among the IAP elements. In the regions, synonymous substitutions occurred more frequently than non-synonymous ones, which suggested that the regions in question were functionally constrained until fairly recently. The rate of nucleotide substitutions in the regions was estimated to be 6-10 X 10(-9) nt per site per year, and significantly higher than that of the cellular genes. These rates may exemplify a characteristic of the nucleotide substitutions for an endogenous retrovirus. The sequence homology between the IAP element and IgE-binding factor gene is discussed.

A sequence homology between the pX genes of HTLV-I/II and the murine IL-3 gene.

Searching the protein sequence database for amino acid sequences homologous to the x-lor sequence in the pX region of human T-cell leukemia virus types I and II (HTLV-I/II), we found that there is a region of 38 amino acids where the murine interleukin 3 (IL-3) sequence has a 40% homology with the x-lor sequence. A statistical analysis shows that this homology is highly significant with a probability of 1.57 X 10(-10). The biological implication of this homology is discussed.

Changes in urinary deoxypyridinoline level and vertebral bone mass in the development of adjuvant-induced arthritis in rats.

This study was designed to determine the effect of bone resorption on the development of generalized osteopenia in adjuvant-induced arthritic rats. Thirty of a total of sixty male SD rats, 6 weeks of age, were injected with killed mycobacterium butyricum suspended in mineral oil into the right hind paw and assigned to six groups of 5 animals each. The other thirty animals served as the age-matched noninjected controls. Animals were sacrificed at 4, 7, 10, 14, 21, and 28 days post-injection after measuring the bilateral hind-paw volumes. Twenty-four-hour urinary samples were obtained before sacrifice and the levels of deoxypyridinoline (D-Pyr) and creatinine (CR) were measured. Plasma intact osteocalcin levels were measured by a sandwich enzyme immunoassay at the start, 14 and 28 days after injection. Bone mineral measurement and histomorphometrical analyses were performed on specimens of the third lumbar vertebral body. On the seventh day after injection, arthritic rats showed significant decreases in the values of bone mineral content (BMC) and density (BMD) when compared to controls. Urinary D-Pyr/Cr ratios, however, did not increase on the seventh day, showing a significant increase on the tenth day after injection. The serum osteocalcin level was significantly reduced on the fourteenth day. The trabecular bone volume (BV/TV) in the arthritic rats showed a significant decrease from the seventh day. The trabecular thickness (Tb.Th) value significantly decreased on the seventh day after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Prednisolone prevents decreases in trabecular bone mass and strength by reducing bone resorption and bone formation defect in adjuvant-induced arthritic rats.

We examined the effects of prednisolone (PSL) administration in normal female Sprague Dawley rats and adjuvant-induced arthritic rats at the age of 6 weeks. Rats were intramuscularly injected with PSL twice a week at doses of 0 (control), 10, 30, 90, or 270 mg/kg body weight (b.w.). In the normal rats, serum osteocalcin level at 14 days and serum carboxyterminal pyridinoline cross-linked telopeptide of type 1 collagen (1CTP) level at 28 days in the 270 mg/kg dose group was lower than the respective value in control animals. The BMC and the trabecular bone formation rate (BFR/BS) of the lumbar body (L-4) in the 270 mg/kg dose group at 14 and 28 days were significantly lower than the values in the control rats. In the arthritic rats, however, serum osteocalcin levels in the PSL-treated groups did not differ compared with arthritic controls. The serum 1CTP levels in all of the PSL-treated groups were significantly reduced at 28 days. The age-dependent increases in the L4 BMC, BMD, and L-3 ultimate compressive load values were maintained. The BFR/BS values in the 90 mg/kg and 270 mg/kg dose groups were significantly higher than those in the arthritic control rats. The trabecular osteoclast number and surface values in all of the PSL-treated groups were significantly lower than the values in arthritic controls. These data demonstrate that PSL administration prevented reduction in bone mass and strength of the lumbar trabecular bone in adjuvant-induced arthritic rats by reducing the increase in bone resorption and the decrease in bone formation at both the local and systemic levels.

Methotrexate maintains bone mass by preventing both a decrease in bone formation and an increase in bone resorption in adjuvant-induced arthritic rats.

We examined the effects of low doses methotrexate (MTX) and indomethacin (IND) on bone mass and turnover in normal male Sprague-Dawley rats and those with adjuvant-induced arthritis. Normal and the adjuvant (heat-killed mycobacterium)-injected rats, 6 weeks of age, were given MTX at daily doses of 0.05, 0.1, or 0.2 mg/kg body weight (BW) or IND at a daily dose of 1.0 mg/kg BW. Rats were killed at the start, or at 14 and 28 days. In normal rats, the administration of these agents did not change the lumbar and femoral BMD values, nor did the serum osteocalcin or urinary deoxypyridinoline (D-Pyr) levels. Lumbar trabecular osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS) were decreased in the rats given IND. In the arthritic rats, the administration of MTX did not prevent an early increase of paw edema in the adjuvant-injected limb, but late inflammatory edema was alleviated in the non-injected limb. However, MTX administration at a dose of 0.1-0.2 mg/kg BW maintained an age-dependent increase in the lumbar and femoral BMD values. While serum osteocalcin levels were decreased and urinary D-Pyr values were increased in the arthritic control rats, these bone markers remained at the levels of the normal rats. Decreases in mineral apposition rate (MAR) and bone formation rate (BFR/BS) and increases in the trabecular Oc.N/BS and Oc.S/BS values were prevented by MTX. While IND almost completely prevented inflammatory paw edema, it did not improve the parameters of bone formation. An increase in osteoclasts was prevented and the osteopenia in the lumbar and the femoral bone was only partially prevented by IND. These data suggest that MTX improves bone mass and turnover in the arthritic rat, in which several cytokines that affect bone cells are involved. An increase in bone resorption may be due to prostaglandins, but bone formation defect was suggested to be due to other cytokines such as IL-1, IL-6, and TNF-alpha in this model.

Green fluorescent protein labeling of cytoskeletal structures--novel targeting approach based on leucine zippers.

Green fluorescent protein (GFP) is a valuable marker for intracellular protein localization. However the fusion of GFP with structural proteins can alter their properties, resulting in a loss of fusion protein localization, decreased GFP fluorescence or both. We describe a novel targeting approach based on noncovalent heterodimerization of GFP and cytoplasmic structural proteins. The formation of structural protein/GFP complexes was mediated by modified leucine zipper protein spacers designed to form high-affinity heterodimers. The complexes localized accurately to specific sites within cells, providing selective fluorescence labeling of subcellular structures such as microfilaments or focal contacts.

Why is there no carbonic anhydrase activity available to fish plasma?

Carbonic anhydrase (CA) is absent from the plasma of vertebrates. In vitro, CA in fish plasma will short-circuit the effect of catecholamines, which is to increase red blood cell (RBC) pH and volume, both of which enhance the affinity of hemoglobin for O2. CA was infused into trout for a period of 6 h and injected after 48 h, during which the animal was submitted to deep hypoxia (PO2=30­35 mmHg; 4.0­4.7 kPa). O2 content, lactate content, catecholamine levels, hematocrit, hemoglobin concentration and pHi were similar to those in the saline-infused control group. In contrast, cell volume was significantly higher and pHe, total CO2 content and organic phosphate levels were significantly lower than in the control group. The concentration of CA was not high enough completely to short-circuit the increase in pHi and red blood cell volume caused by catecholamines. The lower pHe in the CA-infused animals could enhance the activity of the Na+/H+ pump, which would keep the nucleotide triphosphate levels low. pH is a balance between acid loading at the muscle and acid excretion at the gills or the kidneys; we cannot distinguish between which of these resulted in a decrease of plasma pH. In conclusion, CA in plasma did not cause the expected reduction in blood oxygen content but did have a marked effect on plasma total CO2 content.

Cardiovascular effects of adrenomedullin in teleost fishes.

Adrenomedullin is a fifty-two-amino acid polypeptide that was first discovered in pheochromocytoma cells, and later in the normal adrenal medullae, lungs, kidneys, and blood. In mammals, adrenomedullin has vasodepressive effects, mainly by decreasing peripheral vascular resistance. I investigated the effects of adrenomedullin in fish to see if this novel neuropeptide would have an effect in lower vertebrates, or if its actions were limited to the higher vertebrates. Bolus injections of adrenomedullin resulted in a reduction of heart rate and dorsal aortic pressure in the rainbow trout Oncorhynchus mykiss. However, adrenomedullin had no effect in the Atlantic cod, Gadus morhua. The effects of adrenomedullin in trout appear to be due to a direct action on the peripheral vasculature, as pre-treatment of celiac artery strips with tetrodoxin had no effect on the ability of adrenomedullin to relax the strip.