Kinetics of the hydrolysis of synthetic substrates by horse urinary kallikrein and trypsin

The kinetic constants for horse urinary kallikrein and trypsin hydrolysis of BAEE, TAME, bradykinin methyl ester and bradykinyl-Ser-Val-Gin-Val-Ser were determined. The values of the ratio kcat/Km show that (1) kallikrein is catalytically less efficient than trypsin for all the substrates (2) the three esters are equally good substrates for trypsin while horse urinary kallikrein is 100-fold more effective on bradykinin methyl ester than on the other substrates (3) for both enzymes the ester of bradykinin is a better substrate than the tetradecapeptide.     

Development of enzyme biosensor based on trypsin and conductometric thin-film electrodes for protein and artificial substrates determination

ULTRA was established on the 1st April 1990, to consider applications made by registered medical practitioners seeking approval to transplant an organ between 2 living unrelated persons in the United Kingdom.     

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I). Effect of substrates on the fragmentation of subunits by trypsin

It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >/=20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.     

A strategy for designing inhibitors of beta-amyloid toxicity

beta-Amyloid peptide is the major protein component of Alzheimer's plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of beta-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with beta-amyloid, is combined with a disrupting element, which alters beta-amyloid aggregation pathways. The synthesis, biophysical characterization, and biological activity of such an inhibitor is reported. This prototype inhibitor is composed of residues 15-25 of beta-amyloid peptide, designed to function as the recognition element, linked to an oligolysine disrupting element. The inhibitor does not alter the apparent secondary structure of beta-amyloid nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. Evidence for these effects includes changes in fibril morphology and a reduction in thioflavin T fluorescence. In addition to its influence on the physical properties of beta-amyloid aggregates, the inhibitor completely blocks beta-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of beta-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.     

Separation of trypsin and peroxidase by ultrafiltration using crosslinked soybean trypsin inhibitor

The author describes the anatomical findings among the variants of brain arteries in 360 deceased where 160 of them had a hemorrhagic or ischemic stroke, while 200 had no brain diseases. Those cases where strokes were revealed the author found a separation in the posterior part of the circle of Willis--between the carotid and vertebro-basillar systems. This could happen due to an aplasia of one or both posterior connective arteries, or due to the absence of a connecting vessel between an atypically diverging posterior brain artery (posterior trifurcation) and the basillar artery. It is assumed that this circumstance has a definite significance in the pathogenesis of hemorrhages.     

Ultrasensitive fluorogenic substrates for serine proteases

Selective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a k(cat), value of 170 s(-1) and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by >10,000-fold and >100-fold for activated protein C (APC). Seven of these substrates have a k(cat) over 100 s(-1) and three of them have a K(M) below 1 microM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor VIIa/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.     

A framework for designing and implementing community benefit standards

Increasingly, health care professionals and the public are asking questions about the role of the hospital in meeting community need including its not-for-profit tax status. This article reviews the community benefit literature, provides a framework for understanding how a hospital community benefit program was developed, and delineates through a structured case study the lessons learned from this experience. It provides the practitioner with a context in which other hospitals may replicate the program and gives researchers a substantive case study that may be used as the basis for the empirical testing of community benefit models. The authors also outline the many difficult issues faced by a typical community hospital as it attempted to examine and develop additional responses to community need.     

Physicochemical approaches to designing NiAl-based alloys for high-temperature operation

The structure and properties of new-type materials based on light refractory nickel monoaluminide NiAl as a structural material are analytically reviewed. The choice of various alloying systems and structural-phase states of NiAl-based structural materials, including structural materials, is analyzed, and the choice of the processes of production of the materials is grounded, as applied to their composition.     

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.     

RNA-RNA interactions between oligonucleotide substrates for aminoacylation

RNA stem-loop microhelices with helix sequences based on tRNA acceptor stems can be charged with specific amino acids. Experiments were designed to test the possibility that microhelices could laterally associate through complementary loop sequences and thereby bring their attached aminoacyl groups close enough together to form a peptide bond. Computer simulations suggested that formation of such complexes would be sensitive to the number of loop nucleotides needed to span the grooves of the quasi-continuous helix of the intermolecular pseudoknot so formed. These predictions were conformed experimentally by observation of complex formation sensitivity to loop size. Complexes with optimized loop sizes had apparent bimolecular dissociation constants of approximately 100 nM with only three complementary base pairs between the respective loops. Single nucleotide substitutions that disrupted the predicted intermolecular loop-loop base-pairing abolished detectable association. Similarly, placing a gap between the short helix formed by loop-loop pairing and the adjacent acceptor stems also diminished complex formation. These experiments establish an experimental basis for microhelix association for peptide synthesis.     

Conceptual framework for designing a national survey of human exposure

The development and implementation of a National Human Exposure Assessment Survey requires a sound conceptual framework in order to select the population for study, the chemicals of concern, and the media and routes of exposure requiring direct and indirect measurements. A three-level conceptual model is presented within a multidimensional space that provides the basic parameters needed to be considered in the design of such a study. The axis common to all three levels is the duration of exposure. A fundamental need in a national survey is information on environmentally relevant chemicals, the biological mechanisms and health responses, the types of personal contact, the environmental concentrations, the sources, and the populations at risk. Application of the model is appropriate for exposures that can lead to acute or chronic health effects. Five chemicals are used to illustrate the need for multimedia and multiroute exposure analyses of the general population.     

Managing time for greater effectiveness: designing doctor''s ideal day

We cannot rely on geriatricians, internists, and family practitioners alone in the medical community to provide all of the geriatric care. Even though there are alternatives to the use of specialists, we cannot afford to ignore the largest group of current physician trainees who will provide a great deal of geriatric medical care in the future. We need to help make the basic principles of geriatric care part of every training program for every resident, whether in general or specialty programs.     

针对固定重量板坯的板坯设计优化算法

针对板坯重量被事先确定的情况,提出了客户订单重量需求规格和宽度需求规格为区间值的板坯设计问题,建立了最小化板坯数量和总盈余量的多目标板坯设计模型.基于构建的订单-板坯矩阵和板坯相容集合,提出了一种两阶段的板坯设计最优算法,算法的第一阶段实现板坯数量最小化,第二阶段实现总盈余量的最小化.针对提出的算法,给出了最优性质的理论证明以及基于实际数据的应用算例.     

Structural analysis of peptide substrates for mucin-type O-glycosylation

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.     

Rational and combinatorial strategies for designing oligonucleotides targeted to RNA structures

Many RNA structures play a key role in the regulation of gene expression. We designed synthetic oligonucleotides able to interact with folded RNA regions (see Toulmé et al., Biochimie (1996) 78, 663-673, for a review). We have demonstrated that a decanucleotide can form a triple helix with the stem of the hairpin responsible for ribosomal frame-shifting of the gag-pro message of HTLV-I, leading to the inhibition of translation. We have isolated, through an in vitro selection procedure, from a library composed of oligonucleotides with a random part of 30 nucleotides, sequences able to bind to the TAR RNA element of HIV-1 with a dissociation constant of 20-50 nM. The association between the two partners involve non-canonical interactions. This extends the range of potential targets for antisense sequences to functional RNA structures.     

Perioperative nursing orientation. A primer for designing your own program

The orientation program for OR nurses at Mount Sinai Medical Center has been in place for more than two years. Orientees have given consistently favorable evaluations of the program, and many of them have commented that this was the first real OR orientation that they had received in their careers. Staff acceptance of new employees has improved, and our retention rate of new employees is promising. New nurses who have been through the orientation program now can provide staffing in the OR so that more experienced nurses can participate in the RN first assistant training program that we have introduced. A structured orientation program ensures consistency in the information given to new employees. We continue to revise and improve the program based on changes in the workplace and orientees' evaluations. We believe that the time spent designing our program has paid off well in employee satisfaction and retention--in management terms, the return on investment has been good.