Coulombic forces in protein-RNA interactions: binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66

The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the P0, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the P0 and P2 subsites of RNase A is characterized in detail. Lys66 (P0 subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of kcat and Km for poly(C) cleavage were affected by altering the P0 and P2 subsites. The kcat/Km values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the P0 and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the P0 and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the P0 subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the P0 or P2 subsite. The logarithm of Kd is a linear function of the logarithm of [Na+] over the range 0.018 M 相似文献   

A prospective randomized trial of the surgical blood order equation for ordering red cells for total hip arthroplasty patients

BACKGROUND: The majority of crossmatched blood is for surgical patients, and most of it is never transfused. An alternative system for ordering red cell (RBC) units, called the surgical blood order equation (SBOE), which incorporates specific patient variables for surgical patients, has been developed. STUDY DESIGN AND METHODS: A prospective double-blind randomized trial compared the SBOE with the maximal surgical blood order schedule (MSBOS) system for ordering allogeneic RBC units in 60 patients undergoing total hip arthroplasty. Autologous RBCs were available for none of the patients. RESULTS: There were no differences in patient demographic, surgical, or laboratory variables at any time. The median number (range) of allogeneic RBC units ordered was 2 (2-3) for the MSBOS and 0 (0-3) for the SBOE (p<0.0001). The SBOE ordered the correct number of RBC units for 58 percent of patients, while the MSBOS did so for 7 percent (p<0.0001). The SBOE had a lower crossmatch-to-transfusion ratio than the MSBOS (0.83 vs. 4.12). Costs were also lower with the SBOE. CONCLUSION: Incorporation of patient factors in the use of the SBOE system resulted in increased efficiency of blood-ordering practices for total hip arthroplasty.     

Effect of the high-affinity estrogen receptor ligand ICI 182,780 on the rat tibia

We examined the specificity of the steroidal antiestrogen ICI 182,780 (ICI) on bone and reproductive tissues in adult and growing female rats. Using a 1.5-mg/kg dose (s.c.), we evaluated the effects of ICI on the bone, body weight, uterine weight, serum cholesterol, and serum estradiol in either adult and/or growing rats. ICI increased serum estradiol cholesterol in ovary-intact rats, had no effect on uterine weight in ovariectomized rats, and resulted in uterine atrophy in ovary-intact animals comparable with ovariectomy. In contrast, ICI had no effect on body weight. In bone, ICI significantly increased the rate of periosteal bone formation in long bones of growing and mature female rats. In contrast, ICI had no effect on longitudinal bone growth in rapidly growing rats. When ICI was administered to mature rats with or without ovaries, two-factor ANOVA revealed significant interaction (P < or = 0.05) between ovariectomy and ICI treatment for cancellous bone area and labeled bone perimeter. ICI increased skeletal indices of bone turnover in the cancellous bone of ovary-intact rats but reduced these indices of bone turnover in the cancellous bone of ovariectomized rats. The increase in bone turnover was associated with a reduction in cancellous bone area in the ovary-intact rats. A reduction in bone turnover was similarly associated with an increase in bone area in the ICI-treated ovariectomized rats. In summary, ICI exhibited complete estrogen antagonism in cortical and cancellous bone, partial agonism in cancellous bone, and no activity on tibial longitudinal growth rate of growing ovary-intact rats. The effects in adult rats were influenced by circulating levels of estradiol. ICI had no activity on body weight and complete antagonism on uterine weight. These results demonstrate that a ligand with high binding affinity to the estrogen receptor(s) can elicit an array of estrogen-mediated regulation of bone metabolism.     

Ligand-induced conformational change in transferrins: crystal structure of the open form of the N-terminal half-molecule of human transferrin

Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.     

Computed tomography-guided central venous catheter placement in a patient with superior vena cava and inferior vena cava occlusion

Erythropoietin (EPO) is a glycoprotein hormone produced principally by the kidney and is the major stimulus for erythropoiesis. Recombinant human EPO has now been biosynthesized and is available for clinical use, particularly in patients with renal failure. EPO has been reported to be effective in treating anaemia due to chronic renal failure. It has been used in pregnancy to correct anaemia following renal transplantation with graft dysfunction. We report here the case of a post-renal transplant patient who became pregnant and developed severe anaemia which was not related to iron, B12, or folate deficiency. Her anaemia was successfully treated with EPO with no evidence of rejection or significant graft dysfunction following therapy. She tolerated EPO very well, and there was a successful outcome of the pregnancy. This case has encouraged us to conclude that EPO has a useful role in the treatment of anaemia in pregnant women following renal transplantation.     

BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.