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PubMed2006

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples.

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.

Agar↗
PubMed2006

Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water.

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.

Bacteriological Techniques↗
PubMed2004

Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man.

Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae, Plasmodium ovale and Plasmodium vivax have been expressed in recombinant form, and properties of the active site of each enzyme characterized by kinetic analysis. A panel of chromogenic peptide substrates systematically substituted at the P3, P2, P2' and P3' positions was used to estimate enzyme/ligand interactions in the corresponding enzyme subsites based upon kinetic data. The kinetic parameters kcat, Km and kcat/Km were measured to identify optimal substrates for each enzyme and also sequences that were readily cleaved by the plasmepsins but poorly by host aspartic peptidases. Computer generated models were utilized to compare enzyme structures and interpret kinetic results. The orthologous plasmepsins share highly similar subsite specificities. In the S3 and S2 subsites, the plasmepsin 4 orthologs all preferred hydrophobic amino acid residues, Phe or Ile, but rejected charged residues such as Lys or Asp. In S2' and S3' subsites, these plasmepsins tolerated both hydrophobic and hydrophilic residues. Subsite specificities of the plasmepsin 4 family of orthologs are similar to those of human cathepsins D and E, except in S3' where the plasmepsins accept substrates containing Ser significantly better than either of these human aspartic proteases. Peptidomimetic methyleneamino reduced-peptide inhibitors, which have inhibition constants in the picomolar range, were prepared for each plasmepsin 4 ortholog based upon substrate preferences. A peptidomimetic inhibitor designed for plasmepsin 4 from P. falciparum having Ser in P3' had the lowest Ki of the series of inhibitors prepared, but did not significantly improve the selectivity of the inhibitor for plasmepsin 4 versus human cathepsin D.

Animals↗
PubMed2003

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine.

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.

Anticoagulants↗
PubMed2004

A collaborative study to establish a Korean Standard for factor VIII:C concentrate.

BACKGROUND AND OBJECTIVES: A collaborative study among five laboratories including three manufacturers and two national control laboratories was carried out to evaluate the suitability of a candidate to serve as a Korean Standard for factor VIII:C concentrate. MATERIALS AND METHODS: Two approaches were attempted to determine the potency of this candidate. The one is a one-stage clotting assay and the other is a chromogenic assay. To achieve acceptable precision and accuracy, the following recommendations by the International Society on Thrombosis and Haemostasis were adopted in the assays, e.g., pre-dilution of samples in factor VIII (FVIII)-deficient plasma, inclusion of 1% albumin in the dilution buffer and calibration against the sixth International Standard for the blood coagulation factor VIII:C concentrate, coded 97/616. RESULTS: The data collected within each laboratory and among laboratories for both assays employed here were in good agreements in the calibration of the candidate preparation against the International Standard. The overall potencies by the one-stage clotting assay and the chromogenic assay, however, showed recognizable differences between them. Each differed from each other in that the potency obtained from chromogenic assay was approximately 17% lower than that from one-stage clotting assay. The estimated geometric mean value obtained from the one-stage clotting assay was 8.4 international units (IU)/vial and that from the chromogenic assay was 6.7 IU/vial. CONCLUSIONS: Based on the results of this collaborative study, the candidate standard is judged to be suitable to serve as a Korean National Standard for factor VIII:C concentrate.

Biological Assay↗
PubMed2004

Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin.

We have developed an in vitro protocol for the measurement of clot-bound thrombin. This protocol uses a chromogenic substrate for thrombin and a microtiter plate reader and is suitable for screening inhibitors for thrombin that are directed to clot-bound thrombin. Clots were obtained after recalcification of human plasma. For the measurement of clot-bound thrombin, we read the optical density (OD) at 405 nm on a spectrophotometer and compared the results to that obtained with a standard curve of human alpha thrombin. We stopped the amidolytic reaction at 10 min because the optical density was linear until 20 min under our experimental conditions. We suggest that clot-bound thrombin can be measured using a chromogenic substrate specific for thrombin under our experimental condition.

Chromogenic Compounds↗
PubMed2006

Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin generation in vitro.

BACKGROUND: Thrombin generation potential, a critical haemostatic measure, can be determined by continuous detection of total thrombin or direct subsampling. However, differences between methods exist in area under the curve or peak thrombin calculated. Also, impact of anticoagulants on thrombin generation may vary depending on mode of analysis. OBJECTIVE: We studied the effect of components on thrombin generation in the presence or absence of anticoagulants. METHODS: The continuous method was conducted with plasma +/- fibrin(ogen) +/- fibrin polymerization inhibitor. Plasma contained slow-reacting TG5134 substrate at 37 degrees C and reaction was started with dilute thromboplastin in CaCl(2)/Tris buffer. Absorbance (405 nm) was recorded over time and free thrombin calculated from total thrombin activity. For the subsampling method, similar plasma mixtures +/- TG5134 were reacted and free thrombin measured directly as the difference in activity against S2238 substrate of timed subsamples taken into EDTA or EDTA + antithrombin + heparin. RESULTS: Slow-reacting substrate in the continuous method acted as a competitor for thrombin, giving delayed but greater free thrombin than direct subsampling. These differences persisted to varying extents with all anticoagulants tested. In either method, presence of polymerization inhibitor increased the amount of free thrombin. Continuous method detection of alpha(2)macroglobulin complexes was hampered by sensitivity limits leading to inordinate free thrombin calculations. Especially with hirudin, although free thrombin remained at the end of the subsampling method, continuous method calculations assumed no residual free thrombin. CONCLUSION: In vitro plasma thrombin generation is delayed and increased by slow-acting substrate and fibrin polymerization inhibitor.

Adult↗
PubMed2004

Neutralization of venoms from two Southern Pacific Rattlesnakes (Crotalus helleri) with commercial antivenoms and endothermic animal sera.

The Southern Pacific Rattlesnake (Crotalus helleri) is found in southwestern California (USA), southward through north Baja California (MX) into the northern part of southern Baja California (MX). In this study, the venoms from two Southern Pacific Rattlesnakes were characterized. The two venoms were different in color, concentration, and enzyme activities. Two commercial antivenoms neutralized both C. helleri venoms differently. Antivipmyn (Fab2H) and CroFab (FabO) neutralized both venoms but had different ED50. Four times more Fab2H antivenom was required to neutralize the C. helleri venom No. 011-084-009 than the venom from the snake No. 010-367-284. The hemorrhagic activity of two C. helleri venoms were neutralized differently by endothermic animal sera having a natural resistance to hemorrhagic activity of snake venoms. Opossums and Mexican ground squirrel sera did not neutralize the hemorrhagic activity of the venom No. 010-367-284. The sera of gray woodrats and hispid cotton rats neutralized all hemorrhagins in both C. helleri venoms. This is the first reported case in which opossum serum has not neutralized hemorrhagic activity of pit viper venom. Differences in the compositions of C. helleri venoms and their ability to be neutralized may help explain why snakebites are a difficult medical problem to treat and why effective polyvalent antivenoms are difficult to produce.

Animals↗
PubMed2004

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.

Animals↗
PubMed2004

Evaluation of the recurrence of denture stomatitis and Candida colonization in a small group of patients who received itraconazole.

OBJECTIVES: The aim of the study was to determine the recurrence rate of denture stomatitis and persistence of Candida in 22 patients (5 male and 17 female, mean age 71 years) over a 3-year period. STUDY DESIGN: Denture hygiene practice, denture cleanliness, and the presence of palatal erythema were assessed for each patient at the start of the study (baseline). The oral cavity was sampled for yeasts by imprint culture and denture discs. Ten patients received a capsular form of itraconazole (100 mg twice daily for 15 days) and 12 patients were provided with 100 mg of itraconazole in the form of a mouthwash (10 mL twice daily), which was then swallowed. No further antifungal treatment was administered to any of the patients. Clinical and microbiological assessments were repeated for each patient at 6 months and 3 years after the original appointment. Yeasts were identified by colony color on CHROMagar Candida, germ-tube formation, and API-32C profiling. Selected isolates were then typed by inter-repeat polymerase chain reaction (IR PCR). RESULTS: Candida albicans was isolated at baseline from all patients either alone (12 patients) or in combination with another species (10 patients). Other yeast species recovered were C glabrata (5 patients), C tropicalis (1 patient), C guilliermondii (1 patient), C krusei (1 patient), C parapsilosis (1 patient), C kefyr (1 patient), and Saccharomyces cerevisiae (2 patients). Candida albicans and/or C glabrata were recovered from 11 of the 22 patients after 6 months or 3 years. A complete and consistent change of yeast species from baseline was observed in 6 patients after 6 months and at 3 years. The remaining 5 patients were yeast-free at the follow-up assessments. PCR fingerprinting of C albicans and C glabrata indicated strain persistence over 6 months in 10 patients and in 4 patients after 3 years. A switch in strain type occurred for 1 patient after 6 months and for 3 patients after 3 years. CONCLUSIONS: The recurrence of denture stomatitis in patients who maintained a high standard of denture cleanliness was low. Although itraconazole was beneficial in reducing the fungal load, there may be strain persistence or subsequent recolonization of the oral cavity by a broader range of potentially less sensitive yeast species.

Aged↗
PubMed1999

Ultrasonographically guided direct gene transfer in utero: successful induction of beta-galactosidase in a rabbit model.

OBJECTIVE: We sought to determine whether the transfer of enzyme-encoding genes in utero can be detected after birth. STUDY DESIGN: An adenoviral vector carrying the gene for beta-galactosidase was injected under ultrasonographic guidance into the livers of 4 rabbit fetuses per litter (3 litters total) at 27 days' gestation. On delivery of the pups 2 to 3 days later, the livers were analyzed for beta-galactosidase activity by using 5-bromo-4-chloro-3-indolyl-beta-D -galactopyranoside (X-gal) staining. Polymerase chain reaction was also performed on liver extracts as an additional independent measure of successful vector delivery. RESULTS: Successful targeting of the livers of fetal rabbits was demonstrated by beta-galactosidase activity in the nuclei of liver serosal cells, parenchymal hepatocytes, or columnar cells of the gallbladder in 7 (58%) of 12 injected pups and by polymerase chain reaction in liver extracts from 10 (83%) of 12 injected pups. CONCLUSIONS: These results suggest that vectors that carry genes for specific enzymes can be delivered to fetal organs in utero and that expression of the enzyme can be detected after delivery.

Adenoviridae↗
PubMed2000

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

Archives↗
PubMed2002

Novel tools for the study of class I alpha-mannosidases: a chromogenic substrate and a substrate-analog inhibitor.

The use of chromogenic substrates for evaluation of class I alpha-mannosidase is described. 2('),4(')-Dinitrophenyl-alpha-D-mannopyranoside allows rapid and sensitive assays of enzymatic activities, e.g., of heterologously expressed alpha-1,2-mannosidase from Trichoderma reesei. Interaction constants of several ligands with alpha-mannosidases from class I and II could also be determined. Furthermore, novel types of inhibitors derived from D-lyxose are presented. Methyl-alpha-D-lyxopyranosyl-(1(')-->2)-alpha-D-mannopyranoside is a potent inhibitor of the alpha-1,2-mannosidase from T. reesei (K(i)=600 microM) and since it probably spans subsites -1/+1, this disaccharide could be valuable in crystallographic studies of class I alpha-mannosidases.

Binding, Competitive↗
PubMed2002

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates.

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.

Catalysis↗
PubMed2003

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes.

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.

Bacterial Proteins↗
PubMed1999

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

Adenoviridae↗