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Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice., (© 2020 Wiley-VCH GmbH.)

We evaluated the performance of three chromogenic media (BBL CHROMagar™ Staph aureus, ChromID™ S. aureus SAID, ChromID™ S. aureus Elite SAIDE) for the isolation of Staphylococcus aureus in respiratory samples in patients with cystic fibrosis in comparison with CNA media. We reported a similar ability of the four media to support the growth of S. aureus and that sensitivity increased when incubation lasted more than 24 h. SAIDE had the higher sensitivity compared to the other media and kept a high specificity even after 72 h., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

We report the conjugation of a chromogenic cephalosporin β-lactamase (βL) substrate to polymers and integration into biomaterials for facile, visual βL detection. Identification of these bacterial enzymes, which are a leading cause of antibiotic resistance, is critical in the treatment of infectious diseases. The βL substrate polymer conjugate undergoes a clear to deep yellow color change upon incubation with common pathogenic Gram-positive and Gram-negative bacteria species. We have demonstrated the feasibility of formulating hydrogels with the βL substrate covalently tethered to a poly(ethylene glycol) (PEG) polymer matrix, exhibiting a visible color change in the presence of βLs. This approach has the potential to be used in diagnostic biomaterials for point-of-care detection of βL-producing bacteria, helping combat the spread of drug resistant microbes.

Introduction: Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration., Aim: In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods., Methods: Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One-Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma-derived concentrate and recombinant FIX samples were also included for comparison in every assay., Results: Substantial discrepancies were observed when assaying dalcinonacog alfa using the one-stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one-stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma-derived or recombinant samples. However, incubation time influenced in the one-stage clotting assay using Dapttin., Conclusions: Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration., (© 2020 The Authors. Haemophilia published by John Wiley & Sons Ltd.)

Rapid and accurate identification of pathogens involved in urinary tract infections helps to guide antimicrobial therapy. Chromogenic agars provide presumptive identification directly from primary isolation media. They have been intended to make the bacterial isolation and identification process easier and faster. Our study aimed to compare the performance and the cost of the CPS ID3® and the Uriselect4® chromogenic agars with the conventional method for the isolation and identification of urinary tract infections bacteria. We included 301 urinary samples in a prospective study conducted in May 2018 in the clinical laboratory of the National institute of nutrition and food technology of Tunis. Isolates from routine media were identified using API® system while isolates from chromogenic media were directly identified by colony color with reference to the manufacturer's recommendations. Chromogenic media yielded more pure positive cultures and allowed better isolation of Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Morganella morganii and Streptococcus agalactiae. Sensitivity and specificity of the presumptive identification of most commonly isolated uropathogens were higher with the Uriselect4® medium than with the CPS ID3® medium. Chromogenic media yielded the identification of pathogenic organisms 24 hours sooner than the conventional method in approximately 63 % of cases with the CPS ID3® medium and in 77.7% of cases with the Uriselect4® medium. Chromogenic media allowed a much better isolation of bacteria commonly involved in urinary tract infections with a quick, easy and accurate presumptive identification especially with the Uriselect4® medium.

The neuropeptide Y (NPY) Y4 receptor is a G protein coupled receptor, which is targeted by pancreatic polypeptide, a homologue of NPY. Selective Y4R agonists were suggested as potential therapeutics for the treatment of obesity. Highly potent dimeric peptidic Y4R agonists, constituting two pentapeptide moieties connected through an aliphatic linker, represent an interesting class of Y4R ligands. Based on this compound class, photoresponsive Y4R ligands, containing an azobenzene, azopyrazole, diethienylethene or a fulgimide chromophore were prepared to explore structural requirements of such Y4R agonists on Y4R binding. The synthesized Y4R ligands, containing a non-aliphatic rigid photochromic linker, switch reversibly in aqueous buffer and exhibited high Y4R affinity throughout. This demonstrated that the replacement of the highly flexible aliphatic linker by a considerably less flexible photochromic linker was well tolerated with respect to Y4R binding. Differences in Y4R affinity and activity between the individual photoisomers (varying in spatial orientation and flexibility) were marginal suggesting that the linking element in the dimeric ligands is less critical for the adaptation of high-affinity binding modes at the receptor.

Introduction: Tetracycline molecules comprise a group of broad-spectrum antibiotics whose primary mechanism of action is the inhibition of protein synthesis through the binding of the bacterial ribosome. In addition, tetracyclines inhibit matrix metalloproteases (MMPs), a family of zinc-dependent proteases that contribute to tissue remodeling, inflammation, and angiogenesis and are overexpressed in certain pathophysiologies such as diabetic foot ulcers (DFUs)., Objective: This study aims to develop a liquid chromatography and mass spectrometry (LC-MS/MS) doxycycline quantification methodology to facilitate the development of a stable topical doxycycline hyclate (DOXY) formulation as well as evaluate the topical DOXY formulation for the efficacy in MMP-9 inhibition in vitro and in a clinical application of diabetic lower extremity wounds., Materials and Methods: A simple quantification method utilizing LC-MS/MS was used to develop a topical DOXY formulation, a sample of which was analyzed in stability testing. The formulation was evaluated in vitro for MMP-9 activity using a commercial assay and compared with internal kit controls as well as in a clinical setting for wound healing., Results: Two formulations of 2% (w/w) DOXY demonstrated acceptable stability (±10% target concentration) for 70 days when stored at 4°C. Using an in vitro assay of MMP-9 enzyme activity, the 2% DOXY formulation imparted a ~30% decrease in MMP-9 inhibitory potential as compared with the control drug alone (IC₅₀ values 62.92 µM and 48.27 µM, respectively). This topical product was evaluated for clinical utility in a patient with a DFU, and preliminary data suggest this intervention may promote wound healing., Conclusions: In summary, novel DOXY formulations may be stable and biologically active tools amenable to complex wound care.

Enterobacteriaceae are a part of the human intestinal flora easily spread by hand carriage, water and food. Treatment of infections due to Enterobacteriaceae is difficult mainly in countries with a low socioeconomic state. Carbapenems are considered as a last-resort antibiotic for the treatment of infections due to multidrug-resistant Enterobacteriaceae. Carbapenem-resistant Enterobacteriaceae (CRE) presents a major threat to public health and are rapidly disseminating globally. The most important resistance mechanism of Enterobacteriaceae to carbapenems is carbapenemase production. The treatment options for CRE are limited; attention is focused here on the rapid detection of CRE. Laboratory surveillance of cultures and screening of patients are essential and desirable in areas where these strains are endemic. Culture-based methods such as chromogenic media are used for the initial detection of these strains. These media offer a sensitive, convenient, and low-cost way of identifying CRE species. This article provides an overview of the current state of culture-based chromogenic screening media for the detection of CRE., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) causing severe damage of the luminal gastrointestinal tract. Differential diagnosis between both disease entities is sometimes awkward requiring a multifactorial pathway, including clinical and laboratory data, radiological findings, histopathology and endoscopy. Apart from disease diagnosis, endoscopy in IBD plays a major role in prediction of disease severity and extent (i.e. mucosal healing) for tailored patient management and for screening of colitis-associated cancer and its precursor lesions. In this state-of-the-art review, we focus on current applications of endoscopy for diagnosis and surveillance of IBD. Moreover, we will discuss the latest guidelines on surveillance and provide an overview of the most recent developments in the field of endoscopic imaging and IBD., (© 2018 Japan Gastroenterological Endoscopy Society.)

We evaluated the isolation and quantitation of Clostridium difficile from aqueous and fecal samples utilizing ChromID CDIF and cycloserine, cefoxitin, and fructose-containing agar with horse blood and taurocholate media. Growth was similar between the two. ChromID CDIF provided enhanced isolation and required no ethanol pretreatment to inhibit normal flora. ChromID CDIF also improved turn-around time, requiring only 24 hours' incubation., (Copyright © 2015. Published by Elsevier B.V.)

Escherichia coli is the most common bacterial cause of urinary tract infections. Its rapid and specific identification in urine samples represents a major challenge within the rendering results and optimizing the management of the patient. We aimed to compare the sensitivity and specificity of two commercially available chromogenic media for E. coli: ChromID CPS (Biomérieux) and UriSelect4 (Bio(-)Rad), without carrying out further tests. 99 consecutive and non-redundant urine samples considered to be infected were simultaneously plated onto blood agar and the two chromogenic media. Colony color and bacterial growth quantification were compared 18 and 48 hours after incubation. Bacteria were identified with mass spectrometry. A complementary analysis on 80 bacterial strains known to pose potential identification problems was performed. 43 urines samples grew E. coli, and 42 of them were pink-colored on the two chromogenic mediums, as expected (sensibility=97.7%). Growth quantification was significantly greater on blood agar than on chromogenic media (p<0.001).We noted specificity issues at the complementary analysis with the UriSelect4 medium: Citrobacter freundii and some strains of Citrobacter brakii, Enterobacter cloacae and Hafnia alvei were pink-colored, and could be misidentified as E. coli. ChromID CPS medium did not show such misidentification. In conclusion, the agar ChromID CPS proved to be greater than the UriSelect4 agar in our work in terms of specificity of direct identification of E. Coli, without the use of additional test.

With the increasing incidence and diverse etiologies of fungal infections, chromogenic yeast culture media are increasingly used for routine diagnosis. Rhodotorula species, which are characterized by the production of carotenoid pigments, are considered as emerging opportunistic pathogens. We recently diagnosed two fungemia due to Rhodotorula spp. and noticed that in both cases, the yeast failed to grow in subculture on the chromogenic yeast culture medium. This study was thus undertaken to investigate more thoroughly the ability (or inability) of Rhodotorula species to grow on different commercially available chromogenic media for yeast. Eighteen Rhodotorula spp. were checked for their ability to grow on four chromogenic yeast culture media: CHROMagar Candida (BD), Candi 4 Select (Biorad), Brilliance Candida (Oxoid), and Candida ID 2 (BioMerieux). All the Rhodotorula spp. strains grew on Brilliance and Candida ID 2, while only six isolates grew on Candi 4, and seven on CHROMagar. Two chromogenic yeast culture media showed a significant inhibitory effect on the growth of Rhodotorula species. As all Rhodotorula species are resistant to echinocandins and fluconazole, it is essential to isolate and identify these yeast quickly to initiate appropriate amphotericin B antifungal treatment as early as possible. The choice of media for routine use should take into account the ability of different media to allow all emerging fungal pathogens to grow., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation in mouse lungs. In our present study, QD705-COOH and QD705-PEG activated NF-κB and increased monocyte chemotactic protein-1 (MCP-1) expression in macrophages RAW264.7 via MyD88 dependent Toll-like receptor (TLR) signaling pathways. MyD88 is an adapter protein for most TLRs to activate NF-κB. Silencing expression of MyD88 or p65 with siRNA or co-treatment with a NF-κB inhibitor tremendously abolished QD705s-induced NF-κB activity and MCP-1 expression. The involved TLRs might locate either on the cell surface or inside of cells. Co-treatment with a TLR4 inhibitor completely prevented MCP-1 induction by QD705-PEG. Nevertheless, QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented MCP-1 induction. These findings indicate that, depending on their surface modification, OD705s activate MyD88 dependent-TLRs at the surface or inside of the cells, which is an important mechanism for nanoparticles-induced inflammatory responses. But other MyD88-independent pathways may also involve in these responses., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500 cm⁻¹ M⁻¹. This compound was shown to be a substrate for a variety of β-lactamases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To evaluate methicillin-resistant S. aureus (MRSA) detection methods, we compared (a) mannitol salt agar with cefoxitin (MSA-FX), (b) MRSASelect agar (Bio-Rad), (c) MRSA ID (bioMerieuex), and (d) CHROMagar MRSA (BD Diagnostics) as selective media for culturing nasal swab specimens collected from intensive care unit (ICU) patients and healthcare personnel. A total of 99 (17.1%) cases of MRSA were recovered from 578 specimens. Four (5.5%) cases were identified from healthcare personnel and 95 (18.8%) were from ICU patients. The sensitivity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 83.8, 87.9, 80.8, and 84.8% after 18 hr; 92.9, 94.9, 90.9, and 91.9% after 24 hr; and 96.0, 100, 99.0, and 99.0% after 48 hr, respectively. The specificity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 97.7, 99.0, 98.7. and 99.8% after 18 hr; 97.1, 98.5, 98.1, and 99.5% after 24 hr; and 95.2, 97.7, 97.9, and 99.0% after 48 hr, respectively. In conclusion, all four media showed good results after the 24 hr readings, but MRSA ID and CHROMagar MRSA media required readings at 48 hr due to increased sensitivity at this time point.

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

The analysis of heparin/HS saccharides derived from small amounts of tissue or cells is a considerable technical challenge and the development of methods to characterise these carbohydrates has progressed comparatively slowly. A number of procedures have been devised to tag glycans selectively at the reducing end with a group that will enhance the sensitivity of detection and facilitate chromatographic separations. Outlined in this chapter are two useful strategies designed specifically for the analysis of heparin/HS saccharides. The first involves a fluorophore label, Bodipy-FL-hydrazide, which permits highly sensitive (fmol level) detection of saccharides utilising high performance strong anion exchange chromatography. The second facilitates oligosaccharide separation by gel-permeation chromatography and reverse phase high performance ion-pairing chromatography (RP-HPIPC) through the use of a phenylsemicarbazide tag. The latter also serves as an effective mass tag for electrospray mass spectrometry, permitting enhanced analysis of HS saccharides. These methods provide new opportunities for the development of glycomics approaches to study the structure and function of the heparan sulfate family of glycans.

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.

Novel 7-N-(beta-alanyl)aminophenoxazin-3-one salts 27a-d have been synthesized and tested as chromogenic substrates for beta-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the most common respiratory pathogen in patients with cystic fibrosis. The biological results show that 7-N-(beta-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a bright red colour, making it easily distinguishable from the agar background.

In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one-stage assay are more than double than those by two-stage assay. This may be due to the longer incubation times (10-12 min) in the two-stage assay. This study aimed to determine the time course of the activation phase of the two-stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one-stage and two-stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short- or long-incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23-56%, mean 41%) than after 10 min (19-41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21-64%, mean 37%) than with the longer incubation times usually used (13-29%, mean 23%). These time-course experiments have verified that the longer incubation time used in the two-stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.

Epstein-Barr virus (EBV) is an omnipresent human virus causing infectious mononucleosis and EBV associated cancers. Its protease is a possible target for antiviral therapy. We studied its dimerization and enzyme kinetics with two enzyme assays based either on the release of paranitroaniline or 7-amino-4-methylcoumarin from labeled pentapeptide (Ac-KLVQA) substrates. The protease is in a monomer-dimer equilibrium where only dimers are active. In absence of citrate the K(d) is 20 microM and drops to 0.2 microM in presence of 0.5M citrate. Citrate increases additionally the activity of the catalytic sites. The inhibitory constants of different substrate derived peptides and alpha-keto-amide based inhibitors, which have at best a K(i) of 4 microM, have also been evaluated.

Screening specimens were homogenised in saline 0.9% w/v before either direct inoculation or following enrichment in broth on three chromogenic media (MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In total, 102 of 466 specimens yielded MRSA on at least one medium. After incubation for 16-18 h, the sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after 42 h, and 93%, 95%, 79% and not tested, respectively, following broth enrichment. There were significantly more MRSA colonies on MRSA-Select after 16-18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for identifying MRSA based on the colour of colonies after incubation for 16-18 h was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%, respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively, following broth enrichment. The speed of detection (mean time to report a positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of the three media tested following enrichment, the use of an enrichment broth increased the detection rate of MRSA by 16-24%.

Aims: To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women., Methods and Results: The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar., Conclusions: The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples., Significance and Impact of the Study: This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.

The expressional levels of genes in swarmer cells can be determined by a simple method using X-gal-containing semisolid agars and lacZ-fusion transcription reporter strains of the genes concerned. However, X-gal alone inhibited the swarming of Vibrio, regardless of their ability to digest X-gal. Moreover, X-gal inhibited the growth of V. vulnificus containing functional lacZ. These effects of X-gal itself should be carefully considered when trying to determine the expression levels of genes in swarming cells using X-gal-containing semisolid agar.

Topoisomerase IIa is a nucleic enzyme that affects the topological structure of DNA and also is a target for chemotherapy (ie, anthracyclines). In this study, we coevaluated its protein expression with chromosome 17 and gene status. Using tissue microarrays, 40 cases of sporadic, primary endometrial adenocarcinomas, 5 cases of atypical hyperplasia, and 5 cases of benign hyperplasia were obtained and reembedded into two paraffin blocks with a core diameter of 1 mm. Immunohistochemistry combined with chromogenic in situ hybridization was performed in 2 and 5 microm sections, respectively. Finally using a semiautomated Image Analysis System, we evaluated the levels of Nuclear labeling index of topoisomerase IIa expression. Statistical analysis was performed by SPSS version 11.0 software. The results indicate that chromosome 17 instability (aneuploidy in 7/40 cases) and Topo IIa gene deregulation (amplification in 3/40 and deletion in 1/40 cases) are significant genetic events correlated with biologic behavior in endometrial adenocarcinoma. Because protein overexpression was observed in a significant proportion of the tumors (18/40), detection of the specific gene deregulation mechanism is a crucial process for application of targeted chemotherapies, which are characterized by different levels of cardiotoxicity and other serious effects.

In the study presented here, a new chromogenic medium (CPS ID 3; bioMérieux, Marcy l'Etoile, France) was compared to routine media for the isolation, identification and antimicrobial susceptibility testing of bacteria recovered from urine specimens, and a cost analysis was performed. Escherichia coli, Proteeae tribe, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus agalactiae grew on the chromogenic medium as typical differentiated colonies and were accurately identified even in mixed cultures. Although the similarity of colors produced by isolates belonging to the Klebsiella, Enterobacter, Serratia and Citrobacter (KESC) group prevents differentiation among them, members of KESC were easily identified as coliforms. No substantial difference was observed when comparing the results of antimicrobial susceptibility testing performed on colonies selected from reference media versus CPS ID 3. Use of the new medium was associated with a savings of 75% over the conventional methods and the API system. Furthermore, this medium facilitated a remarkable reduction in the laboratory workload and consequently resulted in additional time and cost savings.

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H(2)O(2). Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O(2)), that when dismutated would generate the local increase in H(2)O(2) required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue., (copyright (c) 2004 Wiley Periodicals, Inc.)

Fast voltage-sensitive dyes are widely used to image cardiac electrical activity. Typically, the emission spectrum of these fluorochromes is wavelength shifted with altered membrane potential, but the optical signals obtained also decay with time and are affected by contraction. Ratiometry reduces, but may not fully remove, these artifacts. An alternate approach has been developed in which the time decay in simultaneously acquired short- and long-wavelength signals is characterized nonparametrically and removed. Motion artifact is then identified as the time-varying signal component common to both decay-corrected signals and subtracted. Performance of this subtraction technique was compared with ratiometry for intramural optical signals acquired with a fiber-optic probe in an isolated, Langendorff-perfused pig heart preparation (n = 4) stained with di-4-ANEPPS. Perfusate concentration of 2,3-butanedione monoxime was adjusted (7.5-12.5 mM) to alter contractile activity. Short-wavelength (520-600 nm) and long-wavelength (>600 nm) signals were recorded over 8-16 cardiac cycles at 6 sites across the left ventricular free wall in sinus rhythm and during pacing. A total of 451 such data sets were acquired. Appreciable wall motion was observed in 225 cases, with motion artifact classed as moderate (less than modulation due to action potential) in 187 and substantial (more than modulation due to action potential) in 38. In all cases, subtraction performed as well as, or better than, ratiometry in removing motion artifact and decay. Action potential morphology was recovered more faithfully by subtraction than by ratiometry in 58 of 187 and 31 of 38 cases with moderate and substantial motion artifact, respectively. This novel subtraction approach may therefore provide a means of reducing the concentration of uncoupling agents used in cardiac optical mapping studies.

Purpose: Tie-1 is a transmembrane tyrosine kinase expressed in vascular endothelial cells during angiogenic processes and vasculogenesis. Here we evaluate targeting of rebuilding endothelium with (125)I-labeled Tie-1 monoclonal antibodies (mAbs) in mice., Experimental Design: At first, activity of Tie-1 kinase during reforming of blood vessels was evaluated in melanoma allografts in transgenic mice with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of the Tie-1 promoter gene. Subsequently, in vivo targeting of the healing wound was evaluated with iodinated Tie-1 mAbs in mice, and finally, after confirming the specificities for targeting, we evaluated the biodistribution of Tie-1 mAbs in a melanoma model., Results: Tie-1 mAbs target epithelial skin wounds in mice. Biokinetics of (125)I-Tie-1 mAbs demonstrate a stabilized equilibrium between the blood and wound over 3 days. The accumulation in wound is 21% injected dose/gram (ID/g) and 17% ID/g at 48 h with the two clones of Tie-1 mAbs, 3c4c7 and 10f11g6. Tie-1 promoter is active in the melanoma model in mice. In melanomas, the tumor accumulation is 5% and 4.4% ID/g, and the tumor:liver values are 2.7 and 4.4, respectively, for the two clones of Tie-1 mAbs, 3c4c7 and 10f11g6. The clearance of (125)I-Tie-1 antibodies is slow when compared with that of the iodinated control antibody., Conclusions: Targeting to wound is demonstrated with the Tie-1 mAbs. Accordingly, tumor targeting of melanoma is expertized with the same antibodies.

Branched polyethylenimine (PEI) chains with an average molecular mass of 2 kDa (PEI2) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting PEI2-GNPs conjugates as vectors for the delivery of plasmid DNA into monkey kidney (COS-7) cells in the presence of serum in vitro has been systematically investigated. The transfection efficiencies vary as a function of the PEI/gold molar ratio in the conjugates, with the best one (PEI2-GNPII) being 12 times more potent than the unmodified polycation. This potency can be further doubled by adding amphiphilic N-dodecyl-PEI2 during complex formation with DNA. The resulting ternary complexes are at least 1 order of magnitude more efficient than the 25-kDa PEI, one of the premier polycationic gene-delivery vectors. Importantly, although unmodified PEI2 transfects just 4% of the cells, PEI2-GNPII transfects 25%, and the PEI2-GNPII/dodecyl-PEI2 ternary complex transfects 50% of the cells. The intracellular trafficking of the DNA complexes of these vectors, monitored by transmission electron microscopy, has detected the complexes in the nucleus <1 h after transfection.

The gamma-diketone analogs 1,2-diacetylbenzene (1,2-DAB) and 2,5-hexanedione (2,5-HD), but not the delta-diketone 1,3-diacetylbenzene (1,3-DAB) or the beta-diketone 2,4-hexanedione, induce neuropathological changes in the rodent central and peripheral nervous systems. The molecular targets of these neurotoxic aromatic and aliphatic gamma-diketones, and of their nonneurotoxic structural analogs and ninhydrin, are examined by assessing their differential reactivity with neural and nonneural amino acids and proteins in vitro and in vivo. Whereas 1,2-DAB is chromogenic and forms polymers with amino acids (notably lysine) and proteins (especially lysine-rich proteins), 1,3-DAB lacks these properties. Ninhydrin forms a chromophore without evidence of protein polymerization. 1,2-DAB preferentially targets neurofilament over microtubule protein in vitro and in situ. Based on protein reactivity, 1,2-DAB is three orders of magnitude more reactive than 2,5-HD. Lysine-rich neurofilament protein subunits NF-H and NF-M are more susceptible than lysine-poor NF-L and beta-tubulin to 1,2-DAB. These observations correlate with the development of proximal (1,2-DAB) and distal (2,5-HD) neurofilament-filled axonal swellings and segregated intact microtubules observed during systemic treatment with aromatic and aliphatic gamma-diketones.

Based on the previous report of McCord and co-workers (Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552), the zinc dithiolate active site of alcohol dehydrogenase (ADH) has been studied as a target for cellular oxidants. In the nitrogen monoxide ((*NO)/superoxide (O(2)) system, an equimolar generation of both radicals under peroxynitrite (PN) formation led to rapid inactivation of ADH activity, whereas hydrogen peroxide and ( small middle dot)NO alone reacted too slowly to be of physiological significance. 3-Morpholino sydnonimine inactivated the enzyme with an IC(50) value of 250 nm; the corresponding values for PN, hydrogen peroxide, and (*NO) were 500 nm, 50 microm, and 200 microm. When superoxide was generated at low fluxes by xanthine oxidase, it was quite effective in ADH inactivation (IC(50) (XO) approximately 1 milliunit/ml). All inactivations were accompanied by zinc release and disulfide formation, although no strict correlation was observed. From the two zinc thiolate centers, only the zinc Cys(2)His center released the metal by oxidants. The zinc Cys(4) center was also oxidized, but no second zinc atom could be found with 4-(2-pyridylazo)resorcinol (PAR) as a chelating agent except under denaturing conditions. Surprisingly, the oxidative actions of PN were abolished by a 2-3-fold excess of (*)NO under generation of a nitrosating species, probably dinitrogen trioxide. We conclude that in cellular systems, low fluxes of (*)NO and O(2) generate peroxynitrite at levels effective for zinc thiolate oxidations, facilitated by the nucleophilic nature of the complexed thiolate group. With an excess of (*)NO, the PN actions are blocked, which may explain the antioxidant properties of (*)NO and the mechanism of cellular S-nitrosations.

Hydrolysis of D-valyl-L-leucyl-L-lysine 4-nitroanilide (1), D-valyl-L-leucyl-L-arginine 4-nitroanilide (2), and N alpha-p-tosyl-L-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis-Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis-Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme-substrate complex and a free substrate molecule.

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline. 2. Both AChE and BuChE have been shown to copurify with peptidases. 3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with beta-amyloid protein in Alzheimer brain tissues. 4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis. 5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE. 6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.

Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ-labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo., (Copyright 2001 Wiley-Liss, Inc.)

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium., (Copyright 2000 Wiley-Liss, Inc.)

Objective: To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL., Methods: We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL., Results: Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL., Conclusion: Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.

The location of DNA photodamage within the epidermis is crucial as basal layer cells are the most likely to have carcinogenic potential. We have determined the action spectra for DNA photodamage in different human epidermal layers in situ. Previously unexposed buttock skin was irradiated with 0.5, 1, 2, and 3 minimal erythema doses of monochromatic UVR at 280, 290, 300, 310, 320, 340, and 360 nm. Punch biopsies were taken immediately after exposure and paraffin sections were prepared for immunoperoxidase staining with a monoclonal antibody against thymine dimers that were quantitated by image analysis. Dimers were measured at two basal layer regions, the mid and the upper living epidermis. The slopes of dose-response curves were used to generate four action spectra, all of which had maxima at 300 nm. Dimer action spectra between 300 and 360 nm were independent of epidermal layer, indicating comparable epidermal transmission at these wavelengths. Furthermore, we observed 300 nm-induced dimers in dermal nuclei; however, there was a marked effect of epidermal layer between 280 and 300 nm, showing relatively poor transmission of 280 and 290 nm to the basal layer. These data indicate that solar UVB (approximately 295-320 nm) is more damaging to basal cells than predicted from transmission data obtained from human epidermis ex vivo. The epidermal dimer action spectra were compared with erythema action spectra determined from the same volunteers and ultraviolet radiation sources. Overall, these spectral comparisons suggest that DNA is a major chromophore for erythema in the 280-340 nm region.

Voltage-sensitive dyes and imaging techniques have proved to be indispensable tools for use in in vitro electrophysiological studies. To avoid motion artifacts in optical recordings, electromechanical uncouplers such as 2,3-butanedione monoxime (BDM) are required. In this study, we sought to determine whether the voltage-sensitive dye RH421 had an effect on the contractility of heart muscle, either alone or in the presence of BDM. Ventricular contractility was studied in (i) isolated rat myocytes and (ii) Langendorff-perfused rat hearts under control conditions, and during perfusion with RH421 or RH421 + BDM. The following results were obtained. (i) The amplitude of cell shortening increased progressively from 6.24 +/- 0.64 to 9.95 +/- 1.02 microm during 15 min of superfusion with 5 microM RH421 (n = 11), and further increased to 12.54 +/- 0.97 microm during washout. In seven cells first perfused with 15 mM BDM and then with 15 mM BDM + 5 microM RH421, the amplitude of the cell shortening first decreased from 5.17 +/- 0.51 to 0.41 +/- 0.19 microm, then the amplitude increased to 2.63 +/- 0.25 microm. (ii) Left ventricular pressure (LVP) of the heart (n = 7) was reduced by 15 mM BDM from 60.7 +/- 2.5 to 2.8 +/- 0.5 mmHg (1 mmHg = 133.3 Pa). LVP increased to 12.8 +/- 1.1 mmHg during subsequent perfusion with 10 microM RH421 in the presence of BDM and did not change (LVP = 12.4 +/- 2.4 mmHg) during washout of the dye. Therefore, RH421 increased the contractility of rat hearts and isolated myocytes with and without BDM.

Bacterial endotoxins as contamination of biopreparations have been estimated by chromogenic LAL test. Study on some compounds (aluminium hydroxide, formaldehyde and merthiolate) being components of vaccines showed no effect on the result of LAL test. The level of endotoxins in virus vaccines with the limits defined in producers certificate was adequate, the level of endotoxin was also low in virus vaccines of undefined requirements. The concentration of endotoxin in bacterial vaccines was differentiated. Considering the results of our experiments, as well as the fact, that the requirements for endotoxin contamination of bacterial vaccines are not available it seems necessary to establish the limits for these group of biopreparations.

Three (0140J, C197C and EF20) out of four strains of Streptococcus uberis exhibited high levels of bound plasmin activity following growth in the presence of bovine plasminogen. The remaining strain (C197) bound considerably less plasmin following growth in the same medium. In contrast to the others, this strain was unable to activate bovine plasminogen. Following growth of strain C197 in the presence of bovine plasminogen and a source of plasminogen activator (urokinase or culture filtrate from strain 0140J) high levels of bacterially associated plasmin were detected. None of the strains was able to activate human plasminogen and only trace levels of plasmin activity were detected in association with the S. uberis following growth in the presence of human plasminogen. All strains were able to bind plasmin activity following incubation in the presence of either bovine or human plasmin. However, in each case the level of activity detected following incubation in human plasmin was approximately five-fold less than that observed following incubation with bovine plasmin. None of the strains bound detectable levels of either human or bovine plasminogen. It is concluded that activation of plasminogen is required prior to binding of plasmin by S. uberis.

The oxime derivative 2,3-butanedione monoxime (BDM) is used as an inorganic phosphatase to probe the phosphorylation state of many cellular proteins including the L-type calcium channel in various tissues. We used BDM further to shed light on the controversy surrounding direct phosphorylation of the L-type Ca2+ channel. We employed a recombinant system that utilizes HEK 293 cells expressing wild type and mutant human heart calcium channels. BDM reversibly reduced the calcium channel current induced by expression of the wild type channel in a concentration-dependent manner with an apparent IC50 value of 15.3 mM. Deletion of part of the carboxyl terminus of the alpha 1 subunit, which contains one putative protein kinase A site, or mutating all of the protein kinase A consensus sites of the pore forming subunit, did not significantly change the apparent IC50 value or alter in any other way the blocking effect of BDM on the expressed currents. Our data suggest that BDM produces reversible modifications of the cardiac calcium channel protein leading to an expected reduction in the amplitude of the expressed currents, but the site of action must be different from that of the consensus sites for protein kinase A dependent phosphorylation.

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site".