Towards objective hand hygiene technique assessment: validation of the ultraviolet-dye-based hand-rubbing quality assessment procedure.

Ultraviolet spectrum markers are widely used for hand hygiene quality assessment, although their microbiological validation has not been established. A microbiology-based assessment of the procedure was conducted. Twenty-five artificial hand models underwent initial full contamination, then disinfection with UV-dyed hand-rub solution, digital imaging under UV-light, microbiological sampling and cultivation, and digital imaging of the cultivated flora were performed. Paired images of each hand model were registered by a software tool, then the UV-marked regions were compared with the pathogen-free sites pixel by pixel. Statistical evaluation revealed that the method indicates correctly disinfected areas with 95.05% sensitivity and 98.01% specificity.

Quantitative impact of direct, personal feedback on hand hygiene technique.

This study investigated the effectiveness of targeting hand hygiene technique using a new training device that provides objective, personal and quantitative feedback. One hundred and thirty-six healthcare workers in three Hungarian hospitals participated in a repetitive hand hygiene technique assessment study. Ultraviolet (UV)-labelled hand rub was used at each event, and digital images of the hands were subsequently taken under UV light. Immediate objective visual feedback was given to participants, showing missed areas on their hands. The rate of inadequate hand rubbing reduced from 50% to 15% (P < 0.001). However, maintenance of this reduced rate is likely to require continuous use of the electronic equipment.

An enhanced method for accessory pathway localization in case of Wolff-Parkinson-White syndrome.

This paper presents an analysis of the Arruda accessory pathway localization method for patients suffering from Wolff-Parkinson-White syndrome, with modifications to increase the overall accuracy. The Arruda method was tested on a total of 79 cases, and 91.1% localization performance was reached. After a deeper analysis of each decision point of the Arruda localization method, we considered that the lead aVF was not as relevant as other leads (I, II, III, V1) used. The branch of the decision tree, which evaluates the left ventricle positions, was entirely replaced using different decision criteria based on the same biological parameters. The modified algorithm significantly improves the localization accuracy in the left ventricle, reaching 94.9%. An accurate localization performance of non-invasive methods is relevant because it can enlighten the necessary invasive interventions, and it also reduces the discomfort caused to the patient.

Development of a virtual reality guided diagnostic tool based on magnetic resonance imaging.

Computed tomography (CT) and virtual reality (VR) made it possible to create internal views of the human body without actual penetration. During the last two decades, several endoscopic diagnosis procedures have received virtual counter candidates. This paper presents an own concept of a virtual reality guided diagnostic tool, based on magnetic resonance images representing parallel cross-sections of the investigated organ. A series of image processing methods are proposed for image quality enhancement, accurate segmentation in two dimensions, and three-dimensional reconstruction of detected surfaces. These techniques provide improved accuracy in image segmentation, and thus they represent excellent support for three dimensional imaging. The implemented software system allows interactive navigation within the investigated volume, and provides several facilities to quantify important physical properties including distances, areas, and volumes.

Evaluation of goose serum amyloid a acute phase response by enzyme-linked immunosorbent assay.

Serum amyloid A (SAA) is of interest as the circulating precursor of amyloid A protein, the fibrillar component of AA (secondary) amyloid deposits, and also as an extremely sensitive and rapid major acute phase protein. Serum concentrations of acute phase proteins (APPs) provide valuable information about the diagnosis and prognosis of various diseases, and thus the relevance of APPs for monitoring the health status of domestic animals is widely accepted. More importantly, the measurement of SAA concentration assists in assessing the prognosis in secondary amyloidosis, which is a common disease of geese, affecting an increasing number of animals. In the present study we introduce a highly sensitive goose-specific ELISA method for measuring SAA concentration in goose serum or plasma samples. Samples were taken from geese of the Landes Grey and Hungarian White breeds, which were stimulated for an acute phase reaction by administration of a commercially available fowl cholera vaccine containing inactivated Pasteurella multocida. Strong and characteristically rapid acute phase responses were measured in both breeds, peaking at approximately 24 h after inoculation. The maximum SAA concentration was 1200 microg/ml. At 72 h postinoculation, the concentrations returned to pre-inoculation values. There was significantly (p = 0.004) less intense response in the control groups; however, a very mild increase of SAA levels was detected due to the stress inevitably caused by the sampling procedure.

Brain image segmentation for virtual endoscopy.

This paper presents an algorithm for fuzzy segmentation of MR brain images. Starting from the standard FCM and its bias-corrected version BCFCM algorithm, by splitting up the two major steps of the latter, and by introducing a new factor gamma, the amount of required calculations is considerably reduced. The algorithm provides good-quality segmented 2-D brain slices a very quick way, which makes it an excellent tool to support a virtual brain endoscope.

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Synthesis of and a comparative study on the inhibition of muscle and liver glycogen phosphorylases by epimeric pairs of d-gluco- and d-xylopyranosylidene-spiro-(thio)hydantoins and N-(d-glucopyranosyl) amides.

D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the key step of the syntheses C-(1-bromo-1-deoxy-beta-D-glycopyranosyl)formamides were reacted with cyanate ion to give spiro-hydantoins with a retained configuration at the anomeric center as the major products. On the other hand, thiocyanate ions gave spiro-thiohydantoins with an inverted anomeric carbon as the only products. On the basis of radical inhibition studies, a mechanistic rationale was proposed to explain this unique stereoselectivity and the formation of C-(1-hydroxy-beta-D-glycopyranosyl)formamides as byproducts. Enzyme assays with a and b forms of muscle and liver glycogen phosphorylases showed spiro-hydantoin 12 and spiro-thiohydantoin 14 to be the best and equipotent inhibitors with K(i) values in the low micromolar range. The study of epimeric pairs of D-gluco and D-xylo configurated spiro-hydantoins and N-(D-glucopyranosyl)amides corroborated the role of specific hydrogen bridges in binding the inhibitors to the enzyme.

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Efficient inhibition of muscle and liver glycogen phosphorylases by a new glucopyranosylidene-spiro-thiohydantoin.

Reaction of C-(1-bromo-1-deoxy-beta-glucopyranosyl)formamide 2 with thiocyanate ions was the key step of a short synthesis of D-glucopyanosylidene-spiro-thiohydantoin 7 which proved to be a potent inhibitor of muscle and liver glycogen phosphorylases.

Two mutations in rat trypsin confer resistance against autolysis.

Due to autodigestion the activity of dissolved trypsin successively decreases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysis sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val118 and Lys145-Ser146. Out of these three sites only the first two exist in rat trypsin, an enzyme that has been the target of protein engineering for more than ten years. In this work Lys61 and Arg117 were replaced by Asn via site directed mutagenesis to transform the corresponding peptide bonds to trypsin resistant ones. Kinetic parameters of K61N, R117N and the double mutant K61N/R117N are practically identical with those of the wild-type enzyme. By contrast, the rate of autolysis of each singly-substituted species is substantially slower than with the parent trypsin. In particular, the double mutant shows dramatically increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequence determination. We propose a model to explain why these two positions play a key role in autolysis and how Ca2+ can influence this process. In addition, our in vitro results strongly support the recently proposed model of human hereditary pancreatitis.

Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

The role of disulfide bond C191-C220 in trypsin and chymotrypsin.

Serine proteases of the chymotrypsin family contain three conserved disulfide bonds: C42-C58, C168-C182, and C191-C220. C191-C220 connects the loops around the substrate binding pocket. Using site directed mutagenesis, cysteines of this disulfide bridge were replaced by alanines in trypsin, in chymotrypsin, and in Tr-->Ch-[S1+L1+L2+Y172W], a mutant trypsin with high chymotrypsin like activity. The functional role of this "active site" disulfide was assessed by comparing the catalytic properties of wild-type and mutant enzymes. Its removal from all three proteases caused a decrease in kcat/KM of two to three orders of magnitude, mainly as a consequence of a dramatic increase in KM. The pH dependence of the activity also changed: the rather wide pH optimum, characteristic of the wild-type enzymes (especially trypsin), narrowed since the pKa in the alkaline region shifted downwards. Results show that C191-C220 is necessary for the high activity of both trypsin and chymotrypsin. By contrast, elimination of this disulfide bridge greatly decreased the specificity of trypsin and of Tr-->Ch-[S1+L1+L2+Y172W], but had no significant change on that of chymotrypsin.

Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis.

Ecotin, a homodimer protein of E. coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C-terminal strands that are arranged as a two-stranded antiparallel beta-sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non-specific secondary binding sites. In this study the genes of wild type ecotin and its Met84Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin --> trypsin specificity conversion.

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin-->trypsin specificity conversion.

Inositol 1,4,5-trisphosphate receptor subtypes in adrenal glomerulosa cells.

Expression of the diverse subtypes of inositol 1,4,5-trisphosphate (InsP3) receptor (IP3R) was examined in rat adrenal glomerulosa cells. The polymerase chain reaction products were characterized by means of DNA sequencing and/or restriction enzyme mapping. The predominant subtype expressed is IP3R-1; its alternatively spliced variants containing and lacking segment S1 are present in comparable amounts. The expression level of IP3R-2 is about a quarter that of IP3R-1, whereas IP3R-3 is expressed at a very low level. Sodium depletion, a chronic physiological stimulus of glomerulosa cells, failed to influence the expression of IP3R-1, as measured by competitive polymerase chain reaction, and failed to modify the ratio of the different receptor subtypes, as studied with restriction enzyme mapping.

Chemical synthesis of the pyruvic acetal-containing trisaccharide unit of the species-specific glycopeptidolipid from Mycobacterium avium serovariant 8.

The functionalized, pyruvic acetal-containing haptenic trisaccharide, p-trifluoroacetamidophenyl 6-deoxy-2-O-(3-O-[4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-meth yl- beta-D-glucopyranosyl]-alpha-L-rhamnopyranosyl)-alpha-L-talopyranosid e (19), a component of the glycolipid from Mycobacterium avium serovar 8 was synthesized. For the preparation of the terminal pyruvic acetal-containing unit, benzyl 2-O-benzyl-3-O-methyl-beta-D-glucopyranoside (6) was condensed with methyl 2,2-di(ethylthio)propionate (1) in the presence of SO2Cl2-CF3SO3H catalyst to yield benzyl 2-O-benzyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-beta -D- glucopyranoside (7S), which was then converted into the suitably substituted glycosyl donor 2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-alph a-D- glucopyranosyl trichloroacetimidate (11). The disaccharide glycosyl acceptor p-nitrophenyl endo-3,4-O-benzylidene-6-deoxy-2-O-(2,4-di-O-benzyl-alpha-L-rhamnopyrano syl)- alpha-L-talopyranoside (15) was glycosylated with 11 in the presence of trimethyl trifluoromethanesulfonate to furnish the protected trisaccharide p-nitrophenyl 2-O-(3-O-[2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-m ethyl-beta- D-glucopyranosyl]-2,4-di-O-benzyl-alpha-L-rhamnopyranosyl)-endo-3,4- O-benzylidene-6-deoxy-alpha-L-talopyranoside (16). After deprotection, this gave the spacer-armed unprotected haptenic trisaccharide 19.

Microscopic protonation constants in tobramycin. An NMR and pH study with the aid of partially N-acetylated derivatives.

Three tetra-N-acetyl derivatives and one tri-N-acetyl derivative of tobramycin (1) have been prepared by partial N-acetylation. Comparison of the pKa values, determined by NMR chemical shift titrations and pH titration of the derivatives, with those of unprotected 1 suggests that protonation equilibria at any particular amino group in 1 are not likely to be influenced by those at other sites. pH-Dependent conformational changes in 1 were assessed on the basis of 1H and 13C chemical shift changes in the derivatives.